3RD MIDWEST
SINGLE MOLECULE
WORKSHOP
UNIVERSITY OF ILLINOIS
AT URBANA-CHAMPAIGN
AUGUST 4 - 5, 2014
University of Illinois – Physics Department – 320 Loomis Lab, 1110 W. Green Street –
Urbana-Champaign, Illinois 61801 – 217-333-3393
CONTENTS
ORGANIZERS
Program – 2
Prof. Yann R. Chemla – University of Illinois
Oral presentation abstracts – 5
Dr. Jaya Yodh – University of Illinois
Poster list – 17
Poster presentation abstracts – 20
Management team:
Participants – 45
Angala Meharry – University of Illinois
Maps – 49
Sandra Patterson – University of Illinois
Contact:
VENUE
mwsmw2014@cplc.illinois.edu
Alice Campbell Alumni Center
http://cplc.illinois.edu/workshops/MWSMW2014
601 South Lincoln Avenue
Urbana, IL 61801
http://www.uiaa.org/alumnicenter/contact.html
SPONSORED BY
1
PROGRAM
MONDAY, AUGUST 4, 2014
ALICE CAMPBELL ALUMNI CENTER
8:00 a.m. - 8:45 a.m.
8:45 a.m. - 9:00 a.m.
Registration & refreshments
Welcome
Yann Chemla – University of Illinois at Urbana-Champaign
Keynote lecture: Stephen Kowalczykowski – University of California, Davis
9:00 a.m. - 10:00 a.m.
“Single-Molecule Visualization of Protein-DNA Complexes: Understanding the
Physics and Chemistry of Biology, One Molecule at a Time”
10:00 a.m. - 10:20 a.m.
Coffee break
SESSION I: “Single-Molecule Interactions”
Chair: Yann Chemla – University of Illinois at Urbana-Champaign
10:20 a.m. - 10:40 a.m.
Talk 1: Sanjeevi Sivasankar – Iowa State University
“Conformational Switching in Single Prion Proteins Promotes Oligomerization”
10:40 a.m. - 11:00 a.m.
Talk 2: Charles Schroeder – University of Illinois at Urbana-Champaign
“Direct Observations of TALE Protein Search Dynamics Along DNA”
11:00 a.m. - 11:20 a.m.
Talk 3: Yi Luo – The Ohio State University
“Nucleosomes Accelerate Transcription Factor Dissociation”
11:20 a.m. - 11:40 a.m.
Talk 4: Sujay Ray – Kent State University
“G-quadruplex Formation in Telomeres Enhances POT1/TPP1 Protection Against
RPA Binding”
11:40 a.m. - 12:00 p.m.
Talk 5: Krishna Sigdel – University of Missouri
“Mechanical Insight into Lipid-protein Interactions Using Bee Venom”
12:00 p.m. - 2:30 p.m.
LUNCH & POSTER SESSION I (Posters I-1 to I-27)
SESSION II: “Molecular machines”
Chair: Wei Cheng – University of Michigan
2:30 p.m. - 2:50 p.m.
Talk 6: Peter Cornish – University of Missouri
“Structured mRNA Induces the Ribosome into a Hyper-rotated State”
2:50 p.m. - 3:10 p.m.
Talk 7: Julia Widom – University of Michigan
“Dissecting the Functions of RNA Helicases in Splicing by Single-molecule FRET”
3:10 p.m. - 3:30 p.m.
Talk 8: Maria Spies – University of Iowa
“Single-molecule Studies of FeS-containing DNA Helicases: Kinetics, Conformational
Dynamics and Molecular Mechanisms”
2
PROGRAM - continued
MONDAY, AUGUST 4, 2014
ALICE CAMPBELL ALUMNI CENTER
3:30 p.m. - 3:50 p.m.
Coffee break
3:50 p.m. - 4:10 p.m.
Talk 9: Eric Tomko – Washington University in St. Louis
“ATP- and NTP-dependent Promoter Opening by the Yeast RNAP II Pre-initiation
Complex”
4:10 p.m. - 4:30 p.m.
Talk 10: Alfonso Brenlla – Wayne State University
“Mechanism of Aromatic Carcinogen Bypass by the Y-family Polymerase Dpo4”
4:30 p.m.- 6:30 p.m.
POSTER SESSION II (Posters II-1 to II-25)
7:00 p.m.- 9:00 p.m.
RECEPTION – BREAD COMPANY, 706 S. GOODWIN AVENUE, URBANA
TUESDAY, AUGUST 5, 2014
ALICE CAMPBELL ALUMNI CENTER
8:50 a.m. - 9:00 a.m.
Introduction & announcements
SESSION III: “Single molecules in live cells”
Chair: Maria Spies – University of Iowa
9:00 a.m. - 9:20 a.m.
Talk 11: Kenneth Ritchie – Purdue University
“Mobility of TonB and FepA in the Membranes of E. coli”
9:20 a.m. - 9:40 a.m.
Talk 12: Wenting Li, University of Wisconsin-Madison
“Single Molecule Study of RelA During the Stringent Response in Live E. coli Cells”
9:40 a.m. - 10:00 a.m.
Talk 13: Rudra Kafle, University of Michigan
“Dynamics of Chromosomal DNA in Escherichia coli”
10:00 a.m. - 10:20 a.m.
Coffee break
10:20 a.m. - 10:40 a.m.
Talk 14: Taekjip Ha – University of Illinois at Urbana-Champaign
“Single Molecules and Cellular Mechanics”
10:40 a.m. - 11:00 a.m.
Talk 15: Yan Mei Wang – Washington University in St. Louis
“Single-molecule Investigation of Intraflagellar Transport Mechanisms”
11:00 a.m. - 11:20 a.m.
Talk 16: Qiong Yang – University of Michigan
“From Molecules to Development: Revealing Simple Rules of Biological Clocks”
11:20 a.m. - 11:50 a.m.
11:50 a.m. - 12:00 p.m.
12:00 p.m. - 1:00 p.m.
Business meeting
Poster awards
Lunch
3
PROGRAM - continued
TUESDAY, AUGUST 5, 2014
ALICE CAMPBELL ALUMNI CENTER
SESSION IV: “New methods”
Chair: Taekjip Ha – University of Illinois at Urbana-Champaign
1:00 p.m. - 1:20 p.m.
Talk 17: Wei Cheng – University of Michigan
“Optical Trapping and Multi-parameter Analysis of Single HIV-1 in Culture Media
Reveal the Positive Cooperativity of Envelope Spikes in Mediating Viral Infection”
1:20 p.m. - 1:40 p.m.
Talk 18: Richelle Teeling-Smith – The Ohio State University
“Exploring Dynamics with Single-Molecule Electron Paramagnetic Resonance”
1:40 p.m. - 2:00 p.m.
Talk 19: Randall Goldsmith – University of Wisconsin-Madison
“What to do when the (fluorescent) lights go out: toward single molecule spectroscopy
with optical microresonators”
2:00 p.m. - 2:20 p.m.
Talk 20: Margaret Rodgers – University of Wisconsin-Madison
“A dual-functioning genetic tag for simultaneous isolation and observation of single
fluorescent complexes from whole cell extract”
2:20 p.m. - 2:40 p.m.
Talk 21: Aleksei Aksimentiev – University of Illinois at Urbana-Champaign
“Probing DNA-protein association through atomistic and coarse-grained simulations”
2:40 p.m. - 3:00 p.m.
Talk 22: Michael Hudoba – The Ohio State University
“Design of Force-Sensitive DNA Origami Components”
3:00 p.m.
Closing remarks & departure
4
ORAL PRESENTATION ABSTRACTS
KEYNOTE LECTURE
Single-Molecule Visualization of Protein-DNA Complexes: Understanding the Physics and
Chemistry of Biology, One Molecule at a Time
Stephen Kowalczykowski
University of California, Davis
It is now possible to image individual proteins acting on single molecules of DNA. Such imaging affords
unprecedented interrogation of fundamental biophysical processes. Visualization is achieved through the
application of two complementary procedures. In one, a single DNA molecule is attached to a polystyrene bead,
which is captured in an optical trap. The DNA, a worm-like coil, is extended either by the force of solution flow
in a micro-flow cell, or by capturing the opposite DNA end in a second optical trap. In the second procedure,
DNA is attached by one end to a glass surface. The coiled DNA is elongated either by continuous solution flow or
by subsequently tethering the opposite end to the surface. Proteins and DNA are visualized via fluorescent
reporters. Individual molecules are imaged using either epifluorescence microscopy or total internal reflection
fluorescence (TIRF) microscopy. Molecules are introduced and supramolecular complexes are built, one
component at a time, using microfluidic flowcells. Using these approaches, we have watched proteins functioning
in the repair, replication, and manipulation of DNA. We have imaged unwinding of DNA by helicases,
translocation along DNA by motor proteins, self-assembly of protein filaments on DNA as well as regulation of
nucleation and growth, the search for DNA sequence homology protein-DNA filaments, replication of DNA, and
nucleosome structure and it’s remodeling. I will summarize how these experiments were done, what we’ve
learned, and prospects for the future.
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MONDAY AUGUST 4, 2014
SESSION I: SINGLE-MOLECULE INTERACTIONS
Talk 1: Conformational Switching in Single Prion Proteins Promotes Oligomerization
Sanjeevi Sivasankar
Iowa State University
Transmissible spongiform encephalopathies which include neurodegenerative diseases like Creutzfeldt–Jakob
disease, bovine spongiform encephalopathy, chronic wasting disease and scrapie, are characterized by the
misfolding and oligomerization of prion proteins into protease resistant neurotoxic aggregates. However, the
molecular mechanisms of prion misfolding and aggregation are unclear. Here we report, at the single molecule
level, the role of the structured and disordered regions of prion proteins and different divalent ion co-factors in
their protease resistance and oligomerization. Using a single molecule fluorescence based protease resistance
assay, we demonstrate that prion monomers misfold to a protease resistant conformation before oligomer
assembly; the intrinsically disordered N-terminal region and Cu2+ ions are obligatory for this conformational
switching. Using single molecule force measurements with an Atomic Force Microscope (AFM) we show that the
protease resistant conformation has a 900-fold higher association constant compared to the native conformation.
The high binding affinity of protease resistant prions indicates that they serve as monomeric seeds for the
subsequent formation of neurotoxic aggregates.
Talk 2: Direct Observation of TALE Protein Search Dynamics Along DNA
Charles M. Schroeder
University of Illinois at Urbana-Champaign
In this talk, we discuss the direct observation of transcription activator-like effector (TALE) protein dynamics
along DNA templates using single molecule techniques. TALE proteins are robust, programmable DNA-binding
proteins that can be fused to a nuclease domain to generate the TALEN system, a leading technology in the field
of genetic engineering. In recent years, powerful methods for gene editing have been developed, including zinc
finger nucleases, the CRISPR/Cas9 system, and TALENs. Despite great promise for treating human disease,
however, we still lack a complete understanding of the mechanisms governing TALE search dynamics and the
role of off-target binding events that threaten to inhibit clinical implementation. From this view, our work aims to
develop a molecular-level understanding of TALE binding and target sequence search on DNA, which will
facilitate the design of new and efficient TALEN systems. In this work, we developed a single molecule assay to
directly visualize the binding and 1-D search dynamics of TALE proteins along stretched, dual-tethered DNA
templates. We implemented an efficient method for specific labeling of TALE proteins using an aldehyde-based
bioorthogonal labeling scheme relying on formylglycine generating enzyme. Single molecule data on TALE
search dynamics reveal a previously unknown two-state “search and bind” model, wherein rapid periods of 1-D
diffusion along DNA are interspersed with stagnant periods of protein binding. The two-state kinetic model
reconciles the ability for TALE proteins to quickly locate their target sequence amongst thousands of potential
binding sites. We further generated a series of truncated TALE variants and observed the dynamics of these
proteins at the single molecule level. In this way, we are able to identify the role of TALE subdomains on protein
search, thereby further advancing the understanding of TALE dynamics. Overall, our work provides a “first-ofits-kind” view of the 1-D diffusion of TALE proteins on DNA, which will be critically important for the
engineering of improved TALE proteins for precise genomic editing.
6
Talk 3: Nucleosomes Accelerate Transcription Factor Dissociation
Yi Luo, Justin A. North, Cai Chen, Gary Kash, Sean Rose, Ralf Bundschuh, Michael G. Poirier
The Ohio State University
Transcription factors (TF) bind DNA target sites within promoters to activate gene expression. TFs achieve high
binding specificity on their recognition DNA sequence by binding with resident times of up to hours in vitro.
However, in vivo TFs can exchange on the order of seconds. The factors that regulate TF dynamics in vivo and
increase dissociation rates by orders of magnitude are not known. We investigated TF binding and dissociation
dynamics at their recognition sequence within duplex DNA, single nucleosomes and short nucleosome arrays
with single-molecule Total Internal Reflection Fluorescence (smTIRF) microscopy. We find that the rate of TF
dissociation from its site within either nucleosomes or nucleosome arrays is increased by 1000-fold relative to
duplex DNA, which can be explained by a competitive partial binding model. Our results suggest that TF binding
within chromatin could be responsible for the dramatic increase in TF exchange in vivo. Furthermore, these
studies demonstrate that nucleosomes regulate DNA-protein interactions not only by blocking DNA-protein
binding but by dramatically increasing the dissociation rate of protein complexes from their DNA target sites.
Talk 4: G-quadruplex Formation in Telomeres Enhances POT1/TPPq Protection Against RPA
Binding
Sujay Ray, Ray, Jigar N. Bandaria, Mohammad H. Qureshi, Ahmet Yildiz and Hamza Balci
Kent State University
Human telomeres terminate with a single-stranded 3′ G overhang, which can be recognized as a DNA damage site
by replication protein A (RPA). POT1/ TPP1 heterodimer, a part of the shelterin complex, binds specifically to
single-stranded telomeric DNA (ssTEL) and protects G overhangs against RPA binding. The G overhang
spontaneously folds into various G-quadruplex (GQ) conformations. It remains unclear whether GQ formation
affects the ability of POT1/TPP1 to compete against RPA to access ssTEL. Using single-molecule Förster
resonance energy transfer, we showed that POT1 stably loads to a minimal DNA sequence adjacent to a folded
GQ. At 150 mM K+, POT1 loading unfolds the antiparallel GQ, as the parallel conformation remains folded.
POT1/TPP1 loading blocks RPA’s access to both folded and unfolded telomeres by two orders of magnitude.
This protection is not observed at 150 mM Na+, in which ssTEL forms only a less-stable antiparallel GQ. These
results suggest that GQ formation of telomeric overhangs may contribute to suppression of DNA damage signals.
Talk 5: Mechanical Insight into Lipid-protein Interactions Using Bee Venom
Krishna P Sigdel, Nagaraju Chada, Tina R. Matin, Stephen White, Martin Ulmschneider and Gavin King
University of Missouri
Lipid-protein interactions play vital roles in the stability and activity of membrane proteins. Melittin, a small
linear peptide consisting of 26 amino acid residues, is a major toxic component in the venom of the European bee
Apis mellifera. This cationic peptide is soluble but also has amphiphilic properties which make it suitable for
monitoring lipid-protein interactions in membranes. In this work melittin was functionally and covalently tethered
to an AFM tip and advanced towards a supported lipid bilayer and then retracted from that lipid surface. The goal
of these force spectroscopy experiments was to directly measure the mechanical interaction between melittin and
a POPC lipid bilayer. These measurements shed light upon the structure/function relationship of melittin in a
near-native lipid environment.
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SESSION II: MOLECULAR MACHINES
Talk 6: Structured mRNA Induces the Ribosome into a Hyper-rotated State
Peter Cornish
University of Missouri
During protein synthesis, mRNA and tRNA are moved through the ribosome by the process of translocation. The
small diameter of the mRNA entrance tunnel only permits unstructured mRNA to pass through. However, there
are structured elements within mRNA that present a barrier for translocation that must be unwound. The ribosome
has been shown to unwind RNA in the absence of additional factors, but the mechanism remains unclear. Here,
we show using single molecule Förster resonance energy transfer and small angle X-ray scattering experiments a
new global conformational state of the ribosome. In the presence of the frameshift inducing dnaX hairpin, the
ribosomal subunits are driven into a hyper-rotated state and the L1 stalk is predominantly in an open
conformation. This previously unobserved conformational state provides structural insight into the helicase
activity of the ribosome and may have important implications for understanding the mechanism of reading frame
maintenance.
Talk 7: Dissecting the Functions of RNA Helicases in Splicing by Single-Molecule FRET
Julia R. Widom, Matthew L. Kahlscheuer and Nils G. Walter
University of Michigan
The process of splicing is a critical step in gene expression, and defects in the splicing pathway are associated
with many human genetic diseases. The spliceosome is unlike many macromolecular machines in that it lacks a
pre-formed catalytic core and is built through sequential steps of assembly and rearrangement on the template of
the pre-mRNA. These rearrangement steps require the activity of numerous RNA helicases and ATPases. Prp22
is an RNA helicase found in yeast (with a human homologue, HRH1) that is required for the release of the mRNA
product after the two chemical reactions of splicing are complete. I will present experiments in which singlemolecule FRET is used to investigate the unwinding activity of Prp22 on model substrates, with the goal of
understanding how this unwinding activity is functionally utilized in the spliceosome. I will also describe earlier
experiments that applied smFRET to the study of the entire spliceosome, using the technique of single-molecule
pulldown FRET (SiMPull-FRET) to isolate complexes stalled before the first reaction of splicing. The study of
Prp22 will be extended to the spliceosome using these techniques, which will allow us to isolate from yeast
extract spliceosomal complexes that have been stalled after the second step of splicing by a dominant negative
mutation in Prp22. This will permit investigation of the effects of Prp22 on mRNA dynamics after both steps of
splicing and during mRNA release.
8
Talk 8: Single-molecule Studies of FeS-containing DNA Helicases: Kinetics, Conformational
Dynamics and Molecular Mechanisms
Maria Spies
University of Iowa
DNA helicases are integral components of molecular machines that orchestrate and regulate a broad range of vital
cellular processes including DNA replication, repair, and recombination. XPD is a DNA helicase with a critical
role in nucleotide excision repair (NER). Two biochemical activities of XPD are critical for NER: it separates
duplex DNA at the site of DNA damage and verifies that the damage is indeed NER-reparable. XPD-like
helicases are composed of a Superfamily II motor core and two family-specific auxiliary domains: FeS cluster
domain and ARCH domain. Practical utility of FeS clusters lays in their ability serve as endogenous quenchers of
a wide spectral range of fluorophores. We exploited this phenomenon to characterize helicase-substrate and
helicase-partner interactions as well as protein domain dynamics. I will discuss our findings regarding the
mechanism by which XPD auxiliary domains define translocation polarity, ability to translocate on the proteincoated DNA and to signal the presence of DNA damage. To probe the motions of the XPD auxiliary domains, we
developed a dual illumination single-molecule assay and a data analysis routine which enabled direct
visualization of the binding and dissociation of single, fluorescently labeled DNA molecules while
simultaneously observing changes in the distance between the ARCH and FeS domains. We show that ARCH
domain undergoes thermally driven open-close transitions. The presence of CPD, a prototypical UV lesion,
stabilizes a closed state of the ARCH. Direct access to the microscopic dynamics of XPD revealed how DNA
binding and ARCH domain conformational transitions are connected to kinetically enhance the damage detection
process and to recruit downstream factors in the NER pathway.
Talk 9: ATP- and NTP-dependent Promoter Opening by the Yeast RNAP II Pre-initation
Complex
Eric J. Tomko, James Fishburn, Steven Hahn, and Eric A. Galburt
Washington University in St. Louis
Transcription initiation in Eukaryotes depends on the formation of the multi-protein complex known as the PreInitiation Complex (PIC). In S. cerevisiae, transcription initiation on TATA-dependent promoters has at least four
phases. First, PIC-formation is nucleated by the binding of TATA-box binding protein (TBP) to the TATA-box
element on the promoter. Second, the Ssl2/XPB subunit of TFIIH catalyzes DNA unwinding and establishes a
DNA bubble. Third, the PIC performs transcription-start-site scanning (TSS scanning) where DNA sequences
downstream of the initial site of DNA opening are surveyed for potential initiation sites. Lastly, RNAP II escapes
the PIC and enters into processive transcription elongation. The mechanism of DNA opening and subsequent
transcription-start-site scanning is unknown. Here, using single-molecule magnetic tweezers approaches, we
measure the distributions of DNA bubble size and lifetime formed in the presence of reconstituted PICs with
varying ATP and NTP concentrations on both negatively and positively supercoiled DNA. Our data allow us to
place constraints on the mechanism of transcription initiation in Eukaryotes and serve as a foundation for future
single-molecule studies of this complex and critical step in eukaryotic transcription regulation active site to the
3’-5’ exonuclease site.
9
Talk 10: Mechanism of Aromatic Carcinogen Bypass by the Y-family Polymerase Dpo4
Alfonso Brenlla, David Rueda and Louis J. Romano
Wayne State University
Y-family polymerases such as Dpo4 perform the majority of trans-lesion DNA synthesis in vivo. In this work,
we characterize Dpo4 binding dynamics, conformational rearrangements and catalytic activity in the presence and
absence of carcinogenic DNA adducts. Our single-molecule fluorescence setup enables monitoring polymerase
movement on a DNA substrate with single-nucleotide resolution. We observe that in the absence of nucleotides
the binary complex shuttles between two different conformations within ~1 s. We term these two conformers as
the preinsertion complex, in which the nucleotide-binding site is occupied by the terminal base pair and the
insertion complex, where Dpo4 has translocated one base pair, thus making the dNTP binding site available.
Interestingly, only the preinsertion complex was captured in a crystal structure. Binding of either correct or
incorrect dNTPs result in the formation of an insertion ternary complex. However, if the n+1 template base is
complementary to the incoming dNTP, a structure consistent with a dNTP-stabilized misalignment is observed, in
which the template base at the n position is no longer base paired. In the second part of this work, we used a
DNA template with a single adduct modification corresponding to either 2-aminofluorene (AF) or N-acetyl-2aminofluorene (AAF), two well characterized carcinogenic arylamines. We find that in the absence of nucleotides
both adducts distort polymerase binding, but addition of dNTPs induces the formation of a ternary complex with a
conformation similar to the one observed with a natural DNA substrate. Finally, we observed that
misincorporation pathways for both adducts present significant differences. While AF induces primer-template
slippage, its acetylated counterpart AAF presents a dNTP-stabilized misalignment mechanism.
TUESDAY, AUGUST 5, 2014
SESSION III: SINGLE MOLECULES IN LIVE CELLS
Talk 11: Mobility of TonB and FepA in the Membranes of E. coli
Kenneth Ritchie
Purdue University
Iron uptake is essential for most pro- and eukaryotic cells. Cells attempt to sequester environmental iron, both for
their own use and as a defense against pathogens, by producing iron sequestering proteins such as transferrin,
lactoferrin and ferritin. In response, Gram-negative bacteria chelate iron in the high-affinity siderophore ferric
enterobactin (FeEnt). Transport of FeEnt across the Escherichia coli outer membrane occurs through a TonBdependent process, where it is hypothesized that the inner membrane proteins TonB-ExeBD transfer energy
across the periplasm to the outer membrane iron transporter FepA. Using a green fluorescent protein-TonB
conjugate (TonB-GFP) and labeling FepA by Alexa-555 (A555-FepA), we have monitored the mobility of these
molecules at the single molecule level. Both proteins display strongly confined motion in their respective
membranes. Depolarization of the inner membrane, deletion of ExeBD or depolymerization of cytoskeletal MreB
had minimal effect on the mobility of either protein. Addition of FeEnt modifies the confinement of a fraction of
the TonB to mimic that of FepA, implying interaction in the presence of ligand.
10
Talk 12: Single Molecule Study of RelA during the Stringent Response in Live E. coli Cells
Wentling Li, Heejun Choi, Yan Zhang, Emmanuelle Bouveret, James C. Weisshaar
University of Wisconsin-Madison
The stringent response is a physiological response that occurs when bacterial cells encounter nutritional stresses
such as amino acid starvation or fatty acid starvation. The most marked outcome of this response is an immediate
accumulation of the effector nucleotides, guanosine tetra- and pentaphosphate (ppGpp and pppGpp).The RelA
protein of Escherichia coli is a (p)ppGpp synthetase that is activated by amino acid starvation. Here, we use
single molecule tracking method (sptPALM) to investigate the RelA protein association and dissociation behavior
before and after the stringent response. In contrast to an earlier work in which RelA was found to diffuse like
ribosomes in normal growth conditions and to diffuse freely following the stringent response, we find RelA
diffusion under both conditions to be heterogeneous. And during the stringent response, RelA diffuses more
slowly than in the normal growth condition. Analysis of all single molecule tracking trajectories of RelA by a
hidden Markov model was consistent with two diffusion states where amino acid starvation increases the dwell
time of RelA on the ribosome and promotes the accumulation of RelA in the state with slow mobility. These
observations show that during the stringent response, RelA tends to bind to ribosomes more often compared to the
normal growth condition, suggesting that RelA needs to be “on” ribosomes to synthesize (p)ppGpp.
Talk 13: Dynamics of Chromosomal DNA in Escherichia coli
Rudra P. Kafle, Molly Liebeskind, Thaige Gompa, and Jens-Christian Meiners
University of Michigan
We investigate intracellular dynamics associated with the conformational fluctuations of the chromosomal DNA
in live Escherichia coli cells by Fluorescence Correlation Spectroscopy (FCS). These fluctuations move the
bound fluorophores stochastically into the diffraction-limited excitation volume of a focused laser beam in a
confocal microscope. From the time correlation functions of the measured fluorescence intensity, we quantify the
fluctuations of the DNA as measured by its time-dependent mean square displacement and the viscoelastic
moduli of the nucleoid. These quantities in live cells significantly differ from the ATP-depleted dead cells on
longer time scales, indicating that the fluctuations on longer time scale may be driven by active processes
involving molecular motors that generate forces by ATP hydrolysis. On shorter time scales, we see little
difference between live and dead cells, suggesting that the processes on corresponding short length scales rely
primarily on thermally-driven diffusive mechanisms. We note that the rheological properties of E. coli nucleoid
significantly change when the ATP hydrolysis in cells is inhibited. We compare our results to that of the in vitro
experiments with the concentrated solution of lambda-DNA.
11
Talk 14: Single Molecules and Cellular Mechanics
Taekjip Ha
University of Illinois at Urbana-Champaign
The living cell is an ensemble of molecules that are not individually “alive”, but through their hierarchical
association are capable of sustaining, repairing, and duplicating the cell, rendering it alive. As we aspire to build a
quantitative narrative of the living cell, the next frontier in single-molecule techniques will lie at the interface
between the molecular and cellular. I will describes an approach we are developing in order to bring the clarity
and precision of modern single molecule biophysical approaches to cell biology. Cell-cell and cell-matrix
mechanical interactions through membrane receptors direct a wide range of cellular functions and orchestrate the
development of multicellular organisms. To define the single molecular forces required to activate signaling
through a ligand-receptor bond, we developed the tension gauge tether (TGT) approach in which the ligand is
immobilized to a surface through a rupturable tether before receptor engagement. TGT serves as an autonomous
gauge to restrict the receptor-ligand tension. Using a range of tethers with tunable tension tolerances, we show
that cells apply a universal peak tension of about 40 piconewtons (pN) to single integrin-ligand bonds during
initial adhesion. We find that less than 12 pN is required to activate Notch receptors. TGT can also provide a
defined molecular mechanical cue to regulate cellular functions. Applications of TGT to cell-cell adhesion and
tumor repopulating cells will also be discussed.
Talk 15: Single-molecule Investigation of Intraflagellar Transport Mechanisms
Yan Mei Wang
Wayne State University
In the past decade, flagella/cilia have come to be known as essential sensory organelles for cells. Flagella/cilia
are hair-like surface projections of many eukaryotic cells. They provide motility and sensory functions for the
cells, and defects in cilia cause a growing number of human disorders ranging from polycystic kidney disease to
Bardet-Biedl Syndrome. The growth and signaling functions of flagella/cilia are maintained by intraflagellar
transport (IFT), where kinesin motors move cargo from the cell body into the flagella and dynein motors remove
material from the flagella to the cell body. The proper functioning of this process requires that the IFT machinery,
which in Chlamydomonas is composed of the kinesin-2 and cytoplasmic dynein 1b motors, IFT trains, and
BBSomes, to exchange motors and reorganize these proteins at the flagellar base and tip. How and where these
reorganizations occur remains elusive. We use single-molecule fluorescence imaging methods to study three IFT
reorganization mechanisms in Chlamydomonas: (i) Upon arrival at the flagellar tip, BBSomes, IFT trains, and
dynein motors together dissociate from the microtubules and diffuse along the flagellar membrane for an average
of 2.3 sec before initiating retrograde IFT. This result identifies the flagellar membrane as the site of IFT
machinery reorganization at the flagellar tip, rather than the lumen as suggested previously. (ii) Kinesin motors,
however, remain on the microtubule and dissociate into the flagellar lumen after an average of 1.6 sec. This
result contradicts the long-held model that kinesin exits flagella by IFT, and proposes a new motor switching
mechanism for eukaryotic cells. (iii) All IFT machinery components are organized in the cytoplasm before
entering flagella, rather than recycling within flagella at the flagellar base.
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Talk 16: From Molecules to Development: Revealing Simple Rules of Biological Clocks
Qiong Yang
University of Michigan
Organisms from cyanobacteria through vertebrates make use of biochemical and genetic oscillators to drive
repetitive processes like cell cycle progression and vertebrate somitogenesis. Despite the complexity and diversity
of these oscillators, their core design is thought to be shared. Notably, most of them contain a core positive-plusnegative feedback architecture. Here we use the early embryonic mitotic cycles in Xenopus as a motivating
example and discuss how the positive feedback functions as a bistable switch and the negative feedback as a timedelayed, digital switch (Yang and Ferrell, Nat Cell Biol, 2013; Ferrell, Tsai, and Yang, Cell, 2011). I will next
discuss our ongoing and future research projects on essential biological clocks in early embryos. We employ
mathematical modeling, microfluidic techniques, and optical imaging for a quantitative understanding of selforganizing behaviors of single cells and single molecules during early embryo development. Interested students
are encouraged to contact me (qiongy@umich.edu) and to visit our webpage (www.umich.edu/~qiongy) for more
details.
SESSION IV: NEW METHODS
Talk 17: Optical Trapping and Multiparameter Analysis of Single HIV-1 in Culture Media
Reveal the Positive Cooperativity of Envelope Spikes in Mediating Viral Infection
Wei Cheng
University of Michigan
Optical tweezers use the momentum of photons to trap and manipulate microscopic objects contact-free in three
dimensions. Although this technique has been widely used in biology and nanotechnology to study molecular
motors, biopolymers and nanostructures, direct optical trapping of viruses has been very limited largely due to the
small size of these nanoparticles. Using optical tweezers that can simultaneously resolve two-photon
fluorescence at single-molecule level, here we show that individual HIV-1 can be optically trapped and
manipulated, which allows multi-parameter analysis of single virions in culture fluid under native conditions. We
show that the number of envelope spikes that are essential for viral infection varies widely on the surface of
individual virions. The efficiency of virus infection varied with the envelope content in a distinct sigmoidal
dependence, which revealed a Hill coefficient of 2.9±0.8 (95% confidence). These results suggest that multiple
envelope spikes cooperate on virion surface to mediate HIV-1 infection. Individual virions differ in their ability
to infect host cells as a result of the molecular heterogeneity of spike content. We hypothesize that HIV-1 virions
with high envelope spike content are preferentially transmitted in human populations, consistent with recent
finding (Parrish et al., PNAS 2013) that transmitted founder viruses contained 1.9-fold more envelope per particle
than chronic control viruses (Supported by NIH Director’s New Innovator Award 1DP2OD008693).
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Talk 18: Exploring Dynamics with Single Molecule Electron Paramagnetic Resonance
Richelle M Teeling-Smith, Ezekiel Johnston-Halperin, Michael G. Poirier, P. Chris Hammel
The Ohio State University
Electron paramagnetic resonance (EPR) is an established and powerful tool for studying the dynamics of
biomolecular systems. EPR measurements on bulk biomolecular samples using a commercial X-band
spectrometer provide insight into atomic-scale structure and dynamics of ensembles of biomolecules. Separately,
single molecule measurements of biomolecular systems allow researchers to capture heterogeneous behaviors that
have revealed the molecular mechanisms behind many biological processes. We have merged these two powerful
techniques to perform single molecule EPR. In this experiment, we selectively label single double-stranded DNA
molecules with nitrogen-vacancy (NV) center nanodiamonds and experimentally demonstrate optical detection of
the magnetic resonance of the single NV nanodiamond probe. Changes in the EPR spectrum reveal the dynamics
and the orientation of the attached DNA molecule relative to the applied magnetic field. Using this new
technique, we have successfully measured the first EPR spectrum of a single biomolecule. This research provides
the foundation for an advanced single molecule magnetic resonance approach to studies of complex biomolecular
systems.
Talk 19: What To Do When the (Fluorescent) Lights Go Out: Toward Single Molecule
Spectroscopy with Optical Microresonators
Randall Goldsmith
University of Wisconsin-Madison
Single-particle spectroscopy is a powerful tool for the mechanistic investigations of chemical and biological
dynamics because unsynchronized processes can be directly observed. However, the traditional reliance upon
fluorescence for single-particle measurements limits such investigations to systems where the target is
fluorescent. Ultrahigh-Q optical microresonators offer a way of eliminating the need for fluorescence by enabling
additional sensitive means of interaction with individual particles. Frequently, the interaction, either resonant or
non-resonant, between an adsorbed analyte species and the propagating mode of the resonator can allow sensitive
single-particle detection. We present a two-beam experimental geometry, where the goal is observation of
spectral dynamics of a known target molecule. Our experiment relies on ultrahigh-Q toroidal optical
microresonators as platforms for photothermal spectroscopy. Transitions are optically driven in the particle of
interest, while the thermalization of the excitation energy is detected by the resonator. The relevant heat flows are
explored with numerical simulations. We demonstrate implementation of the concept by making measurements
on individual carbon nanotubes. Future augmentation to enable spectroscopy on individual non-fluorescent
molecules will be discussed.
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Talk 20: A Dual-functioning Genetic Tag for Simultaneous Isolation and Observation of
Single Fluorescent Complexes from Whole Cell Extract
Margaret L. Rodgers, Joshua Paulson and Aaron A. Hoskins
University of Wisconsin-Madison
Single-molecule colocalization methods excel in detecting and analyzing biomolecular complex formation. Since
large complexes often cannot be reconstituted from purified systems, these studies are often done in cell extracts.
We have developed a new variation of the SiMPull approach1 to isolate and visualize native complexes using a
single genetically incorporated tag. Our SNAP-SiMPull tag utilizes a SNAP tag for fluorophore conjugation fused
to an E. coli biotin acceptor peptide, which can be biotinylated in vivo. Tagged proteins can be pulled down from
cell extract directly onto a streptavidin-coated single-molecule slide. Using this method, we have isolated a
number of complexes involved in yeast pre-mRNA splicing including both U1 and U6 snRNPs comprising 17
and 8 proteins respectively. By introducing multiple fluorescent labels, we have analyzed protein stoichiometry in
these isolated complexes. Finally, we have performed functional assays on the immobilized particles to study
their interactions with other components of the RNA splicing machinery. We predict that this approach will be
broadly applicable for studies of other macromolecular machines.
1. Jain, A. et al. Probing cellular protein complexes using single-molecule pull-down. Nature 473, 484–488
(2011).
Talk 21: Probing DNA-protein Association Through Atomistic and Coarse-grained
Simulations
Aleksei Aksimentiev
University of Illinois at Urbana-Champaign
The fundamental role played by DNA in biology mandates that its sequence be preserved by an organism
throughout its life cycle and be reproduced exactly in its progeny. Experimental studies have already identified all
components of the DNA replication and repair machinery for several model organisms. Furthermore, the structure
and function of the individual components have been characterized in extensive detail. However, just as knowing
the specifications of all players in a team-sport is not sufficient to predict the outcome of the game, knowing the
structure and function of all components of the DNA replication and repair machinery is not sufficient to describe
how the actual process of DNA replication and repair proceeds. In collaboration with single molecule
experimentalists at UIUC, we have been developing a physics-based model to quantitatively describe the
behavior of protein-DNA systems as it emerges from the intricate interactions of the system’s components. We
have designed our model to satisfy the conflicting requirements of being able to handle very large systems at
physiological times scale while retaining the atomic level of details in description of interactions between DNA
and proteins. In this talk, I will describe the applications of our model to studies of DNA systems under
mechanical stress and interactions of single stranded DNA with single-stranded DNA binding proteins of the
DNA replication machinery.
15
Talk 22: Design of Force-Sensitive DNA Origami Components
Michael W. Hudoba, Michael Poirier and Carlos E. Castro
The Ohio State University
Scaffolded DNA origami is powerful design and fabrication tool for the creation of nanoscale objects via bottom
up self-assembly. These objects have ~nm level geometric complexity and spatial accuracy, which is comparable
to biological machinery. DNA origami has been used to create different a wide range of objects such as drug
delivery containers or platforms to guide molecular robots. Current applications of DNA origami exploit the large
stiffness of bundles of dsDNA to create structures that maintain a well-defined and static geometry. However,
DNA origami nanostructures with mechanically functional components, such as springs or actuators have
remained largely unexplored. We aim to make DNA origami devices that are responsive to force magnitudes
typically seen in biomolecular system (~picoNewtons). We have currently developed two approaches to make
force-sensitive DNA origami components. The first (analog approach) uses curved bundles of DNA ‘beams’ that
act as springs to continually measure forces. Conceptually, these springs function similar to a macroscopic leaf
spring, where the equilibrium configuration is curved, and changing the curvature results in a restoring force. This
approach can be used to design springs that deform continuously under an applied force. The second (binary
approach) incorporates structures similar to DNA hairpins into DNA origami designs. The hairpin-like structures
undergo a conformational change at a specific force threshold that can be tuned according to the design. These
two approaches to create force sensitive components are demonstrated through the design of two sensors: an
analog force sensor, and a binary force sensor. These devices are currently being calibrated with the use of
magnetic tweezers, single molecule fluorescence, and flow-based techniques. Ultimately we aim to use these
devices to measure forces of molecular interactions in cellular systems, for example cellular traction forces
applied during cell migration.
16
POSTER LIST
POSTER SESSION I
I-1
Polyamine Mediates Sequence and Methylation-dependent Chromatin Compaction
Jejoong Yoo, Hajin Kim, Taekjip Ha and Aleksei Aksimentiev
I-2
Constructing an Energy Landscape for the Hybridization of Short Oligonucleotides
Kevin Whitley, Matthew J. Comstock and Yann R. Chemla
I-3
Force-dependent Melting of Supercoiled DNA at Thermophilic Temperatures.
Eric Tomko, James Fishburn, Steven Hahn, Eric Galburt
I-4
A Single Molecule Perspective of Sequence Dependence Elasticity in DNA
Julia T. Bourg, Krishnan Raghunathan, Alan Kandinov, Joshua Milstein and Jens-Christian Meiners
I-5
G-quadruplex Conformation and Dynamics Are Determined by Loop Length and Sequence
Ramreddy Tippana, Weikun Xiao, Sua Myong
I-6
Probing the Effect of Different Ligands on the Conformational Dynamics of a Transcriptionally Acting
preQ1 Riboswitch using Single Molecule FRET Microscopy
Jiarui Wang, Krishna Chaitanya Suddala, and Nils Walter
I-7
Single Molecule Study of Repair-specific Functions of Replication Protein A
Ran Chen, David Beyer, Maria Spies, and Marc S. Wold
I-8
Diffusion of Human Replication Protein A along Single Stranded DNA
Binh Nguyen, Joshua Sokoloski, Roberto Galletto, Elliot L. Elson, Marc S. Wold, and Timothy M.
Lohman
I-9
Mechanical Activation of the Hha/H-NS Protein Complex to Condense DNA
Haowei Wang, Samuel Yoshua, Sabrina S. Ali, William W. Navarre and Joshua N. Milstein
I-10
Transcription Factor Binding inside Nucleosomes is Controlled by Core Histone PTMs
Matthew Brehove, Yi Luo and Michael Poirier
I-11
DNA Sequence and Modifications Control Nucleosome Mechanical Stability
Thuy Ngo, Qiucen Zhang and Taekjip Ha
I-12
The Influence of linker histones on transcription factor binding within nucleosomes.
Morgan Bernier, Kingsley Nwokelo, Pei Zhang, Mark Parthun and Michael Poirier
I-13
Quantifying the role of steric constraints in nucleosome positioning
Tomas Rube and Jun Song
I-14
The Influence of Histone H3 with Trimethylated Lysine 36 on the Stability of the Nucleosome
Matthew D. Gibson, Jovylyn Gatchalian, Catherin A. Musselman, Justin A. North, Tatiana G. Kutateladze
and Michael G. Poirier
I-15
Gas-Liquid Phase Transitions in Sub Cellular Level Molecular Mechanisms by Single Molecule FRET
Younghoon Kim, Christian Eckmann, Clifford P. Brangwynne and Sua Myong
I-16
Bloom Helicase Unfolds G-quadruplex in the Absence of ATP
Jagat B. Budhathoki, Sujay Ray, Vaclav Urban, Pavel Janscak, Jaya G. Yodh and Hamza Balci
I-17
RTEL1 Binds and Remodels G-quadruplex DNA
David Beyer and Maria Spies
I-18
The Effect of Single-stranded DNA Binding Protein RPA2 on XPD Helicase Processivity
Barbara Stekas, Zhi Qi, Masayoshi Honda, Maria Spies and Yann R. Chemla
I-19
Single-Molecule Measurement of Mtr4 RNA Helicase Activity Using High-Resolution Optical Trapping
Eric M. Patrick, Sukanya Srinivasan, Eckhard Jankowsky, and Matthew J. Comstock
17
I-20
Single-molecule Imaging Reveals the Translocation Dynamics of Hepatitis C Virus NS3 Helicase
Chang-Ting Lin, Felix Tritschler, Kyung Suk Lee, Meigang Gu, Charles M. Rice and Taekjip Ha
I-21
Differential mechanism of unwinding trinucleotide repeat by yeast Srs2 and Sgs1
Yupeng Qiu, Hengyao Niu, Patrick Sung and Sua Myong
I-22
Investigating the hyper-rotated state of the ribosome, and its possible involvement in frameshifting
Bassem Shebl and Peter Cornish
I-23
Correlation of the L1 Stalk Motion and Subunit Rotation
Drew E. Menke and Peter V. Cornish
I-24
Single molecule FRET study of the Dpo4 behavior at DNA containing Benzo[a]pyrene adduct
Pramodha Liyanage, David Rueda and Louis Romano
I-25
The spliceosome transiently undocks the splicing substrate to promote catalysis, proofreading, and
alternative splicing
Daniel Semlow, Mario Blanco, Nils Walter and Jonathan Staley
I-26
Intramolecular head to tail distance in 2D reveals that kinesin walks with its cargo upright
Kai Wen Teng, Marco Tjioe, Carol S. Bookwalter, Kathleen M. Trybus and Paul R. Selvin
I-27
Characterization of Kinesin-1 Motor Domain Residues Important for Localization within Neurons
Michael Kelliher, Aaron Hoskins and Jill Wildonger
POSTER SESSION II
II-1
Folding Dynamics of a WW Domain Protein Using Single Molecule Force Spectroscopy
Miles L. Whitmore, Dena Izadi, Lisa J. Lapidus and Matthew J. Comstock
II-2
Resolving Prion Protein Aggregation at the Single Molecule Level
Chi-Fu Yen, Anumantha Kanthasamy and Sanjeevi Sivasankar
II-3
Resolving the Molecular Mechanism of Cadherin Catch Bond Formation
Kristine Manibog, Hui Li, Sabyasachi Rakshith and Sanjeevi Sivasankar
II-4
Resolving the Molecular Mechanism of Cadherin Ideal Bond Formation
Sunae Kim, Kristine Manibog, Sabyasachi Rakshith and Sanjeevi Sivasankar
II-5
Characterizing the Interaction of Desmosomal Cadherins at Single Molecule Level
Omer M. Shafraz, Sabyasachi Rakshith, Molly Lowndes, W. James Nelson and Sanjeevi Sivasankar
II-6
Multiple Conformations of a Single SNAREpin between Two Nanodisc Membranes Reveal Diverse
Pre-fusion States
Jaeil Shin, Xiaochu Lou, Dae-Hyuk Kweon and Yeon-Kyun Shin
II-7
Dynamics of the General Secretory System Viewed in Near-native Conditions Via Atomic Force
Microscopy
Raghavendar Reddy Sanaganna Gari, N.C. Frey, L.L. Randall and Gavin M. King
II-8
The Synaptotagmin 1 Linker May Function as an Electrostatic Zipper That Opens for Docking but
Closes for Fusion Pore Opening
Xiaochu Lou, Ying Lai, Yongseok Jho, Tae-Young Yoon and Yeon-Kyun Shin
II-9
Real Time Observation of Lipid-Protein Interactions in Crude Cell Lysates and with Single-Molecule
Resolution
Edwin Arauz, Vasudha Aggarwal, Ankur Jain, Taekjip Ha, and Jie Chen
18
II-10 Single-molecule Studies of Membrane Proteins on Glass Substrates Using Atomic Force Microscopy
Nagaraju Chada, Krishna P. Sigdel, Tina R. Matin, Raghavendar Reddy Sanganna Gari, Chunfeng
Mao, Linda L. Randall, and Gavin M. King
II-11 Cellular Pathways for Productive HIV-1 Entry and Molecular Mechanisms of its Inhibition
Hanna Song and Wei Cheng
II-12 Cell Geometry Affects the Distribution of Measured Diffusion Coefficients in Bacteria
David J. Rowland and Julie S. Biteen
II-13 Spatiotemporal Dissection of Cytoplasmic and Nuclear miRNA Function
Laurie Heinicke, Sethuramasundaram Pichiaya, Elizabeth Cameron and Nils Walter
II-14 Single-molecule Fluorescence Imaging of RecO Localization and Dynamics in Bacillus subtilis
Hannah H. Tuson, Yi Liao, Lyle A. Simmons and Julie S. Biteen
II-15 Single-molecule Mechano-memory
Isaac T.S. Li, Taekjip Ha, and Yann R. Chemla
II-16 Single-molecule Tracking of Elongation Factor P in Live E. coli Cells
Heejun Choi, Sonisilpa Mahapatra, Suparna Sanyal and James C. Weisshaar
II-17 The Princess and the Pea: A Story of Cell Mechanics
Mehdi Roeimpeikar, Qian Xu and Taekjip Ha
II-18 Somitogenesis in Zebrafish
Zhengda Li, Ye Guan and Qiong Yang
II-19 Virion Immobilization Post Diffusion Limits HIV-1 Infectivity Revealed by Real-time Single Particle
Tracking
Michael C. DeSantis, Jin H. Kim, Jamie L. Austin, and Wei Cheng
II-20 Electrical Current Measurement and Manipulation of Single Geobacter Cells Via Optical Trapping
Jess L. West, Adam J Gros, Rebecca J Steidl, Gemma Reguera and Matthew J Comstock
II-21 Optical Trapping and Characterization of Single HIV-1 in Culture Media
Yuanjie Pang and Wei Cheng
II-22 Combined Multi-Color Fluorescence and Ultra-High Resolution Optical Tweezers
Cho-Ying Chuang, Miles L Whitmore, Jess L West, and Matthew J Comstock
II-23 Three Dimensional Localization of Single Biomolecules with Nanometer Resolution
Patrick D. Schmidt, Chi Fu Yen, John Lajoie and Sanjeevi Sivasankar
II-24 Site-specific Labeled HIV-1 Neutralization Antibodies for Single-molecule Fluorescence Measurement
Jin H. Kim and Wei Cheng
II-25 Mechanical Modulation of Enzyme Activity by Dynamic DNA Tweezers
Soma Dhakal, Minghui Liu, Matthew R. Adendorff, Mark Bathe, Hao Yan and Nils G. Walter
19
POSTER PRESENTATION ABSTRACTS
POSTER SESSION I
Poster I-1: Polyamine Mediates Sequence and Methylation-dependent Chromatin Compaction
Jejoong Yoo, Hajin Kim, Taekjip Ha and Aleksei Aksimentiev
University of Illinois at Urbana-Champaign
All cellular activities, including cell development and differentiation, are consequences of gene regulations. In the
conventional view, transcription factors directly regulate gene expressions by activating or repressing the binding
of RNA polymerase to specific target genes. However, a more general and global mechanism of gene regulations
based on compaction level of chromatin is becoming accepted thanks to high-throughput and high-resolution
experimental techniques revealing the chromatin conformations in molecular details. For example, AT-rich
heterochromatin segments of human genome are known to cluster one another forming compact spatial
topologically associated domains (TADs) whereas GC-rich euchromatin segments form separate relatively less
compact TADs. More surprisingly, high level of DNA methylation results in compaction of a specific locus or an
entire chromatin, enabling reversible regulation of gene expressions. Although the correlation between the
chromatin compaction and genomic sequence (GC and methylation contents) is well established, the underlying
principle is poorly understood. Here, we demonstrate that the differential compaction of DNA by sequence can
occur only in the presence of sub-mM polyamine using highly optimized computer simulations and novel singlemolecule techniques, suggesting that a rather simple physical principle of polyamine-mediated DNA-DNA
attractions might govern the global chromatin compaction in the cells. Consistent to the chromatin compaction in
vivo, we find that polyamine-mediated DNA-DNA attraction is significantly stronger for AT-rich and
methylation-rich DNA segments than GC-rich segments. Further, we show that this sequence-dependent DNA
attraction originates from the DNA structure encoded by sequence in atomistic details.
Poster I-2: Constructing an Energy Landscape for the Hybridization of Short Oligonucleotides
Kevin Whitley, Matthew J. Comstock and Yann R. Chemla
University of Illinois at Urbana-Champaign
The hybridization of short oligonucleotides plays a critical role in many biological systems, from DNA
replication to gene silencing. Despite extensive studies, details on the mechanism of this process on the shortest
length scale (~10 bp) remain poorly understood. We use high-resolution optical tweezers with simultaneous
fluorescence microscopy to investigate the hybridization of single oligonucleotides under tension. We measure
the change in end-to-end extension upon annealing and melting as well as the unbinding kinetics of short (7-12
bp), fluorescently labeled oligonucleotides of DNA and RNA hybridizing to a complementary DNA sequence
tethered between trapped beads. Our results allow us to construct an energy landscape of oligonucleotide
hybridization along a well-defined reaction coordinate. Interestingly, our measurements of DNA duplex and
RNA-DNA melting as a function of oligonucleotide length indicate that the transition state for both has the same
end-to-end extension as a ~6 bp duplex. Based on these results and prior studies, we propose that melting occurs
through a common transition state in which the two complementary strands are partially aligned to each other
with a minimal base-paired “core.” Additionally, we find that the change in extension upon hybridization
deviates from extensible wormlike-chain model at forces >10 pN. We propose a model combining shear
deformation and fraying of the terminal base pairs of the oligonucleotides to explain these results.
20
Poster I-3: Force-dependent Melting of Supercoiled DNA at Thermophilic Temperatures
Eric Tomko, James Fishburn, Steven Hahn and Eric Galburt
Washington University in St. Louis
Local DNA opening plays an important role in DNA metabolism as the double-helix must be melted before the
information contained within may be accessed. Cells finely tune the torsional state of their genomes to strike a
balance between stability and accessibility. For example, while mesophilic life forms maintain negatively
superhelical genomes, thermophilic life forms use unique mechanisms to maintain relaxed or even positively
supercoiled genomes. Here, we use a single-molecule magnetic tweezers approach to quantify the forcedependent equilibrium between DNA melting and supercoiling at high temperatures populated by Thermophiles.
We show that negatively supercoiled DNA denatures at 0.5 pN lower tension at thermophilic vs. mesophilic
temperatures. This work demonstrates the ability to monitor DNA supercoiling at high temperature and opens the
possibility to perform magnetic tweezers assays on thermophilic systems. The data allow for an estimation of the
relative energies of base-pairing and DNA bending as a function of temperature and support speculation as to
different general mechanisms of DNA opening in different environments.
Poster I-4: A Single Molecule Perspective of Sequence Dependence Elasticity in DNA
Julia T. Bourg, Krishnan Raghunathan, Alan Kandinov, Joshua N. Milstein and Jens-Christian Meiners
University of Michigan
DNA looping is a ubiquitous and vital regulatory process found in all organisms to maintain proper function and
viability. In protein-mediated looping, the DNA’s sequence dictates both protein binding sites and the local
biomechanical properties. These mechanical properties, notably its elasticity and intrinsic curvature, govern the
ease at which loops can form or breakdown. To probe the effects of sequence dependence on elasticity and the
loop formation process, single molecule experiments were performed on short segments (~150bp) of DNA with
varying amounts of AT and GC content between the protein-binding operators. Tethered particle motion (TPM)
microscopy was employed to observe protein-mediated DNA loop formation in this system, and an analytical
model of the loop formation process was used to calculate the elasticity of the DNA from the observed loop
formation rates. Axial constant-force optical tweezers were used to directly stretch the same DNA molecules
mechanically to determine their persistence length as a measure for their elasticity. Our results indicate that the
intraoperator sequence has a larger effect on elasticity in the loop formation experiments than in the stretching
experiments, which we attribute to different elasticity regimes when the DNA is strongly bent as in a DNA loop,
compared to the thermally induced small curvature fluctuations in stretched DNA.
21
Poster I-5: G-quadruplex Conformation and Dynamics Are Determined by Loop length and
Sequence
Ramreddy Tippana, Weikun Xiao and Sua Myong
University of Illinois at Urbana-Champaign
The quadruplex forming G-rich sequences are unevenly distributed throughout the human genome. Their
enrichment in oncogenic promoters and telomeres has generated interest in targeting G-quadruplex (GQ) for an
anticancer therapy. Here, we present a quantitative analysis on the conformations and dynamics of GQ forming
sequences measured by single molecule fluorescence. Additionally, we relate these properties to GQ targeting
ligands and G4 resolvase 1 (G4R1) protein binding. Our result shows that both the loop (non-G components)
length and sequence contribute to the conformation of the GQ. Real time single molecule traces reveal that the
folding dynamics also depend on the loop composition. We demonstrate that GQ-stabilizing small molecules, Nmethyl mesoporphyrin IX (NMM), its analog, NMP and the G4R1 protein bind selectively to the parallel GQ
conformation. Our findings point to the complexity of GQ folding governed by the loop length and sequence and
how the GQ conformation determines the small molecule and protein binding propensity.
Poster I-6: Probing the Effect of Different Ligands on the Conformational Dynamics of a
Transcriptionally Acting preQ1 Riboswitch using Single-Molecule FRET Microscopy
Jiarui Wang, Krishna Chaitanya Suddala and Nils Walter
University of Michigan
Non-coding RNAs play crucial roles in a multitude of biological processes such as translation, messenger RNA
(mRNA) splicing and regulation of gene expression. Riboswitches are regulatory elements found mainly in the 5’
untranslated regions of numerous bacterial mRNAs capable of modulating gene expression in response to the
binding of cellular metabolites. Riboswitches comprise two functional components: a ligand binding aptamer
domain and an expression platform whose conformation decides the fate of gene expression. Despite its smallest
size among all reported aptamers, the Bacillus subtilis (Bsu) preQ1 (pre-queuosine) riboswitch boasts precise
control of the expression of genes involved in the biosynthesis of queuosine. The co-crystal structure of the Bsu
aptamer domain bound to its ligand preQ1 showed a pseudoknot structure. However, the nature of folding
pathway remained elusive, until recently. We have recently used single molecule fluorescence resonance energy
transfer (smFRET) and computational simulations to show that the ligand-free Bsu aptamer adopts a pre-folded
conformation in which the single stranded A-rich tail interacts with stem-loop P1-L1. Previous studies indicated
that the Bsu riboswitch binds closely related ligands preQ0 and guanine with similarly favorable affinities. The
effects of these ligands on the conformational dynamics, however, were not previously probed and can reveal to
what extent near-cognate ligands stabilize the folded state to affect gene regulation. Here, we use smFRET to
study the effects of preQ0 and guanine in comparison to preQ1 on the kinetics of Bsu aptamer folding. Our
preliminary data show that at saturating concentration and in the presence of Mg2+, guanine stabilizes the folded
state as potently as preQ0. However, surprisingly, in the absence of Mg2+, guanine tends to stabilize the folded
state more efficiently than preQ0. We anticipate this comparative study to elucidate the ligand-mediated folding
pathway of the Bsu aptamer.
22
Poster I-7: Single Molecule Study of Repair-specific Functions of Replication Protein A
Ran Chen, David Beyer, Maria Spies and Marc S. Wold
University of Iowa
Replication Protein A (RPA), the major eukaryotic single-strand DNA (ssDNA) binding protein, is essential for
replication, repair, recombination, and cell cycle progression. Defects in RPA activities lead to genomic
instability, a major contributor to the development of cancer and other diseases. The large subunit of RPA
(RPA1) mediates binding to ssDNA. The RPA-DNA interface contains a series of polar residues and four
conserved aromatic residues. We have used a combination of biochemical analysis in vitro and knockdownreplacement studies in vivo to characterize the contribution of these aromatic residues to RPA function. Mutation
of these aromatic residues results in a separation-of-function phenotype. Cells expressing the aromatic mutants
supported DNA replication, had normal checkpoint activation after DNA damage but were defective in DNA
repair and accumulated double-strand breaks. Also, the aromatic residue mutants were unable to support
nucleotide excision or double strand DNA break repair. We are using single molecule total internal fluorescence
microscopy (smTIRF) and ensemble assays to determine the affinity and kinetics of binding to different DNA
structures including single strand intermediates found at sites of damage and replication. Mutation of the aromatic
residues altered the stability of the RPA-DNA complex and decreased the affinity for short ssDNA regions. Our
results show that DNA replication and DNA repair require different RPA-DNA interactions and that functions in
repair depend on the high affinity DNA-binding domains of RPA1. These studies are contributing to
understanding how human cells maintain genome integrity.
Poster I-8: Diffusion of Human Replication Protein A along Single Stranded DNA
Binh Nguyen, Joshua Sokoloski, Roberto Galletto, Elliot L. Elson, Marc S. Wold and Timothy Lohman
Washington University in St. Louis
Replication Protein A (RPA) is a eukaryotic single stranded (ss) DNA binding protein that plays critical roles in
most aspects of genome maintenance, including replication, recombination and repair. RPA binds ssDNA with
high affinity, destabilizes DNA secondary structure and facilitates binding of other proteins to ssDNA. However,
RPA must be removed from or redistributed along ssDNA during these processes. To probe the dynamics of
RPA-DNA interactions, we combined ensemble and single molecule fluorescence approaches to examine human
RPA diffusion along ssDNA and find that an hRPA hetero-trimer can diffuse rapidly along ssDNA. Diffusion of
hRPA is functional in that it provides the mechanism by which hRPA can transiently disrupt DNA hairpins by
diffusing in from ssDNA regions adjacent to the DNA hairpin. hRPA diffusion was also monitored by the
fluctuations in fluorescence intensity of a Cy3 fluorophore attached to the end of ssDNA. Using a novel method
to calibrate the Cy3 fluorescence intensity as a function of hRPA position on the ssDNA, we estimate a onedimensional diffusion coefficient of hRPA on ssDNA of D1 ~5000 nucleotide2s-1 at 37˚C. Diffusion of hRPA
while bound to ssDNA enables it to be readily repositioned to allow other proteins access to ssDNA. This work
was supported in part by NIH grants GM030498 (T.M.L.), GM044721 (M.S.W.), GM098509 (R.G.) and
HL109505 (E.L.L.).
23
Poster I-9: Mechanical Activation of the Hha/H-NS Protein Complex to Condense DNA
Haowei Wang, Samuel Yoshua, Sabrina S. Ali, William W. Navarre and Joshua N. Milstein
University of Toronto
The bacterial chromosome must be under varying levels of mechanical stress due to a high degree of crowding
and repeated protein-DNA interactions experienced within the nucleoid. DNA tension is difficult to measure in
cells and it is not known if its effects have any functional significance. However, in vitro experiments have
implicated a range of biomechanical phenomena for DNA. The histone-like nucleoid structuring protein, H-NS, is
a key regulator of DNA condensation and gene expression in enterobacteria and is affected by a variety of
cofactors with which it may form a complex, such as the protein Hha. By combining tethered particle motion
(TPM) and optical tweezers experiments we probed the effects of tension on DNA in the presence of the Hha/HNS complex. We find that a brief fluctuation in DNA tension, induced by optical tweezers, causes the rapid and
irreversible compaction of DNA when in the presence of H-NS and Hha. Our results imply that the Hha/H-NS
complex may selectively condense bacterial DNA based upon the level of mechanical tension that is experienced
along different regions of the chromosome.
Poster I-10: Transcription Factor Binding inside Nucleosomes Is Controlled by Histone PTMs
Matthew S. Brehove, Yi Luo and Michael Poirier
The Ohio State University
Genomic DNA in a cell is stored wrapped around a histone octamer core 147 base pairs at a time into repeating
units called nucleosomes. DNA wrapped in nucleosomes is generally inaccessible to DNA binding proteins and
thus nucleosomes control access to the genome. Post translational modifications (PTM’s) to these histones serve
as epigenetic marks that can signal for transcription, repression, and repair. Some of these PTM’s are on the
histone-DNA interface and can alter the accessibility of transcription factors to the nucleosomal DNA. Here our
smFRET data shows the effect of these PTMs on transcription factor binding inside of a nucleosome using
mutants that mimic common modifications.
Poster I-11: DNA Sequence and Modifications Control Nucleosome Mechanical Stability
Thuy T.M. Ngo, Qiucen Zhang and Taekjip Ha
University of Illinois at Urbana-Champaign
Understanding the physical basis of how DNA sequence and modifications affect nucleosome dynamics and
nucleosomal DNA exposure will help elucidate how genomic and epigenetic modifications regulate cellular
functions, cell differentiation and cancer development. Here, we used single-molecule force fluorescence
spectroscopy and single-molecule DNA cyclization measurement to investigate local conformational dynamics of
the nucleosome under tension and its modulation by DNA sequence and modifications. First, we observed that the
nucleosome can unwrap asymmetrically and directionally under force. Second, we showed that nucleosome
mechanical stability is controlled by local DNA flexibility. We demonstrated the correlation between DNA
flexibility and unwrapping force by varying DNA sequence, DNA methylation and DNA mismatches. DNA
methylation decreases DNA flexibility and reduces nucleosome mechanical stability while DNA mismatches
have opposite effects. Our work elucidates the fundamental physical principles for regulation role of DNA
sequence and modifications on nucleosome mechanical and directional stability which controls nucleosome
accessibility for replication, transcription, repair and remodeling.
24
Poster I-12: The Influence of Linker Histones on Transcription Factor Binding Within
Nucleosomes
Morgan W. Bernier, Kingsley Nwokelo, Pei Zhang, Mark Parthun and Michael Poirier
The Ohio State University
The Linker Histone, H1, is involved in the compaction of chromatin into a higher order structure. This
compacted structure inhibits DNA accessibility and thus regulates gene expression. While core histones have
been shown to reduce transcription factor binding, certain post translational modifications of core histones in the
entry-exit region of nucleosomes tend to increase nucleosome unwrapping thus allowing transcription factors to
bind more easily. The effect of H1 on this binding is not well known particularly in the presence of core histone
modifications. Additionally, H1 has several isoforms whose various functions are also not understood. Using a
combination of ensemble and smFRET assays, we explore the binding of H1 and its isoforms and how they
inhibit the binding of the transcription factor, Gal4, within nucleosomes. We also present data on how post
translational modifications contribute to H1’s effect on Gal4 binding. Because of the extremely low Kd of Gal4,
ensemble assays are not enough to observe how H1 affects binding rates. The smFRET studies allow us to
observe individual binding events of both H1 and Gal4 and provide us with rate constants of the interaction
between the proteins and nucleosomes.
Poster I-13: Quantifying the Role of Steric Constraints in Nucleosome Positioning
Tomas Rube and Jun Song
University of Illinois at Urbana-Champaign
Statistical positioning, the localization of nucleosomes packed against a fixed barrier, is conjectured to explain the
array of well-positioned nucleosomes at the 5' end of genes, but the extent and precise implications of statistical
positioning in vivo are unclear. We examine this hypothesis quantitatively and generalize the idea to include
moving barriers as well as nucleosomes actively packed against a barrier. Early experiments noted a similarity
between the nucleosome profile aligned and averaged across genes and that predicted by statistical positioning;
however, we demonstrate that aligning random nucleosomes also generates the same profile, calling the previous
interpretation into question. New rigorous results reformulate statistical positioning as predictions on the variance
structure of nucleosome locations in individual genes. In particular, a quantity termed the variance gradient,
describing the change in variance between adjacent nucleosomes, is tested against recent high-throughput
nucleosome sequencing data. Constant variance gradients provide support for generalized statistical positioning in
50% of long genes. Genes that deviate from predictions have high nucleosome turnover and cell-to-cell gene
expression variability. The observed variance gradient suggests an effective nucleosome size of 158 bp, instead of
the commonly perceived 147 bp. Our analyses thus clarify the role of statistical positioning in vivo.
25
Poster I-14: The Influence of Histone H3 with Trimethylated Lysine 36 on the Stability of the
Nucleosome
Matthew D. Gibson, Jovylyn Gatchalian, Catherin A. Musselman, Justin A. North, Tatiana G. Kutateladze and
Michael Poirier
The Ohio State University
The fundamental unit of chromatin, the nucleosome, consists of DNA wrapped around a histone protein octamer
core. Histones contain a large number of post translational modifications (PTMs), which often function as protein
binding sites. The idea of a histone code has emerged which attempts to link modifications to biological functions.
These PTMs are typically thought to work by directly or indirectly recruiting transcription factors (TFs). However,
proteins which bind these PTMs can also mechanically alter nucleosome dynamics to increase DNA accessibility.
Using Fluorescence Resonance Energy Transfer we investigated the influence of PHF1, which specifically binds
trimethylated Lysine 36 on histone H3 (H3K36me3), on TF affinity to its target sequence buried within
nucleosomes. We find that PHF1 serves to increase TF access to nucleosomal DNA. We measure the affinity
increase due to PHF1s binding domain as about a factor of 3, and find that the domains of PHF1 contribute
multiplicatively. These results suggest that PTM H3K36me3 may serve to facilitate transcription by increasing
DNA accessibility. We plan to expand these studies to single molecule measurements which will elucidate the
effect of binding on TF rates and compaction of nucleosome arrays.
Poster I-15: Gas-Liquid Phase Transitions in Sub Cellular Level Molecular Mechanisms by
Single Molecule FRET
Younghoon Kim, Christian Eckman, Clifford P. Brangwynne and Sua Myong
University of Illinois at Urbana-Champaign
In germ cell development, ribonucleoprotein (RNP) complex termed p-body plays a critical role in mRNA
storage, splicing, degradation and translation repression. Many proteins within p-body contain RNA binding
domains and low complexity (LC) sequences of unknown function. We employed single molecule fluorescence to
characterize LAF1 helicase of C. elegans as a model system to investigate p-body assembly process. LAF-1 is a
DEAD box helicase which possess N-terminal RGG box and C-terminal poly-glutamine tract and a helicase core.
Our results reveal that LAF-1 specifically binds single strand (ss) RNA and induces unexpected compaction and
dynamics of the RNA strand. LAF-1 displays no unwinding of double stranded RNA, yet it promotes annealing
of complementary ssRNA. Series of truncation mutants reveal that the N-terminal RGG box is responsible for
providing the dynamic interaction and RNA annealing whereas other domains contribute to the compaction of
RNA and oligmerization. Native gel analysis indicates that LAF-1 and the mutants form higher order structure,
reflecting their inherent propensity to oligomerize. Together, we unravel the molecular mechanism how LAF-1
may contribute to gas-liquid transition-like behavior of p-granule.
26
Poster I-16: Bloom Helicase Unfolds G-quadruplex in the Absence of ATP
Jagat B. Budhathoki , Sujay Ray, Vaclav Urban, Pavel janscak, Jaya G. Yodh and Hamza Balci
Kent State University
RecQ family helicases, such as BLM and WRN, have been shown to unfold G-quadruplex (GQ)structures in the
presence of ATP. Eliminating BLM and WRN have been shown to cause genomic instability, retardation of
replication machinery, and DNA breaks in potent1ially GQ forming sites of the genome. In addition to helicases,
single strand DNA (ssDNA) binding proteins have also been shown to possess GQ unfolding activity, which
indicates the significance of unfolding these structures for cellular proper functioning of metabolic activities such
as replication, transcription and repair. Motivated by efficient unfolding of GQ by ssDNA binding proteins, we
studied GQ-helicase interactions in the absence of ATP in order to probe possible GQ destabilization due to
binding of a protein in the vicinity of GQ. Using single molecule Förster Resonance Energy Transfer (smFRET)
and bulk biochemical assays, we found that binding of BLM, WRN, and RECQ5 to an ssDNA overhang in the
vicinity of GQ leads to varying degrees of GQ destabilization, including unfolding, in the absence of ATP. We
also observed that the efficiency of BLM-mediated GQ unfolding correlates with the binding stability of BLM to
the ssDNA overhang, as modulated by the nucleotide state, ionic conditions and overhang length. Furthermore,
increasing GQ stability, via shorter loops or higher ionic strength reduces BLM-mediated GQ unfolding.
Poster I-17: RTEL1 Binds and Remodels G-Quadruplex DNA
David Beyer and Maria Spies
University of Iowa
The inactivation of tumor suppressor genes commonly occurs at chromosomal fragile sites, often-repetitive
regions of the genome characterized primarily by a tendency to form secondary structures that impede essential
interactions between the replisome and template DNA. Similarly, the repeated TTAGGG sequence arrays that
compose the human telomere can readily form G-quadruplex (G4) secondary structures in the lagging strand that
can stall replication and lead to drastic changes in telomere stability and length. Many fragile sites across the
human genome have the capacity to form G4-like structures. Essential human DNA helicase RTEL1 co-localizes
with the replisome during telomeric replication and prevents the global generation of fragile sites. As is the case
with most human DNA helicases, RTEL1 likely has several cofactors that target and tune its general biochemical
activities to confer a specific adaptive advantage. It is currently unclear whether RTEL1 prevents the
accumulation of both genomic and telomeric fragile sites by assisting the replisome through difficult structures or
via some other mechanism. We have developed a set of total internal reflection fluorescence (TIRF) assays to
explore interactions between RTEL1 and G4 DNA. Our system employs a biotinylated full-length helicase
construct purified directly from human cell culture. Fluorescence trajectories of RTEL1 incubated with G4 DNA
constructs in the presence of ATP show binding and remodeling behavior. Studies performed in concert with
likely cofactors may clarify the role these behaviors play in a cellular context. Mapping the interaction network
between RTEL1 and both replisomal and telomeric cofactors will elucidate the mechanism of RTEL1’s
demonstrated role in promoting gainful, robust cell cycle progression.
27
Poster I-18: The Effect of Single-stranded DNA Binding Protein RPA2 on XPD Helicase
Processivity
Barbara Stekas, Zhi Qi, Masayoshi Honda, Maria Spies and Yann R. Chemla
University of Illinois at Urbana-Champaign
FacXPD helicase is the archaeal homolog of yeast Rad3 and human xeroderma pigmentosum group D protein
(XPD) from the organism Ferroplasma acidarmanus. This enzyme serves as a model for understanding the
molecular mechanism of human superfamily 2B helicase XPD involved in transcription initiation and nucleotide
excision repair. Previous work has shown that the unwinding of double-stranded DNA by FacXPD is regulated by
the single-stranded DNA binding protein FacRPA2. The mechanism by which this occurs is unknown. Here, we
present a single molecule study of this regulation using combined optical traps and fluorescence. We show that
XPD is a weak helicase as a monomer, only able to unwind short distances (~12 bp) under tension applied by the
optical traps, with a strong dependence on DNA sequence. In the presence of RPA2, however, XPD monomers
are able to unwind more processively (>90 bp). We show that RPA2 on its own can melt ~6 bp of DNA under
tension, suggesting that RPA2 may assist XPD unwinding by binding ahead of the helicase and disrupting the
duplex ahead. Alternately, RPA2 may form a complex with XPD, activating it for processive unwinding. To
distinguish these possible mechanisms, we use a combination optical trap and FRET assay to correlate the
binding of RPA2 with processive unwinding events by XPD.
Poster I-19: Single-Molecule Measurement of Mtr4 RNA Helicase Activity Using HighResolution Optical Trapping
Eric M. Patrick, Sukanya Srinivasan, Eckhard Jankowsky and Matthew J. Comstock
Michigan State University
We present single-molecule high resolution optical trapping measurements of the Mtr4 helicase unwinding an
RNA duplex. RNA helicases act as RNA structure remodelers and play key roles during RNA metabolism, from
transcription, native structure adoption to degradation. During aberrant RNA degradation, the exosome works in
conjunction with other protein factors to get rid of misfolded RNA molecules. These protein factors include
helicases such as Mtr4 that ensure that the RNA to be degraded has the correct structure. To elucidate how Mtr4
unwinds such RNAs, we have performed high resolution optical trapping experiments utilizing a 16-base pair
duplex RNA with an adenosine-rich 3´ end (which has sequence homology to its natural substrate) between two
1.5 kb dsDNA handles tethering a pair of trapped beads. We find that Mtr4 unwinds these RNA duplexes in one
or two step bursts with a mean step size of 9 base pairs. The unwinding is irreversible suggesting that Mtr4
remains stably bound after unwinding the duplex, preventing re-annealing. Closely linked to Mtr4 are two
additional proteins Trf4, which introduces adenosine tags to the target RNA, and Air 1/2, which together make
the TRAMP complex. Future experiments utilizing combined fluorescence and optical trapping measurements
may reveal how Mtr4 activity is coordinated within the TRAMP complex.
28
Poster I-20: Single-molecule Imaging Reveals the Translocation Dynamics of Hepatitis C
Virus NS3 Helicase
Chang-Ting Lin, Felix Tritschler, Kyung Suk Lee, Meigang Gu, Charles M. Rice and Taekjip Ha
University of Illinois at Urbana-Champaign
Worldwide, over 185 million people are chronically infected with hepatitis C virus (HCV), facing risks of
developing liver diseases, such as hepatocarcinoma. There is no vaccine available. New therapies without side
effects are highly needed. HCV encodes a superfamily 2 helicase (NS3h) in the C-terminal portion of
nonstructural protein 3. This enzyme is essential for virus replication. Previous studies have well characterized
the unwinding properties of the helicase. The ensemble approaches, however, have largely limited the
understanding of the translocation dynamics. Here, we used optical traps to stretch kilobase-size single-stranded
DNA (ssDNA), the single-molecule tracking of fluorescence-labeled NS3 enables us to directly determine the
translocation speed, processivity, binding duration and the stoichiometry of translocating complex. Interestingly,
we observed NS3h-mediated repetitive looping of ssDNA in the range of hundreds nucleotides. We further
applied single-molecule fluorescence resonance energy transfer (smFRET) to the analysis of repetitive looping
behavior. By tuning the fluorophore pair position between protein and nucleic acids, more structural information
has been revealed. The dual-ways of movements observed by single molecule analysis may play roles in HCV life
cycle.
Poster I-21: Differential Mechanism of Unwinding Trinucleotide Repeat by Yeast Srs2 and
Sgs1
Yupeng Qiu, Hengyao Niu, Patrick Sung and Sua Myong
University of Illinois at Urbana-Champaign
Trinucleotide repeat (TNR) expansion causes many known inherited neurological and muscular disorders in
human including Huntington’s disease and Friedreich’s ataxia. One source of expansion is the replication defect
at TNR sequence that leads to the hairpin formation and subsequent lengthening of the TNR segment. The Srs2
and Sgs1 are two helicases in yeast that display varying degree of resolving TNR hairpin during replication to
prevent expansion. Using single molecule fluorescence, we investigated the unwinding mechanism by which Srs2
and Sgs1 resolve TNR hairpin and compared it to unwinding of duplex DNA. While Sgs1 unwinds both
structures indiscriminately, Srs2 displays a repetitive unfolding of TNR hairpin without fully unwinding it.
Further, the repetitive unfolding of Srs2 shows dependence on the hairpin folding strength and the total length of
TNR. Our results reveal an exquisite mechanism of Srs2 that may contribute to efficient resolving of the TNR
hairpin.
29
Poster I-22: Investigating the Hyper-rotated State of the Ribosome, and Its Possible
Involvement in Frameshifting
Bassem Shebl and Peter Cornish
University of Missouri
Ribosomes play a pivotal role in the central dogma, translating encoded mRNA to functional proteins. Yet, the
underlying dynamics of translation is not fully understood. We observed a novel translocation intermediate; the
hyper-rotated state (HRS), of the ribosome.1 Along with other local and global conformational changes of the
ribosome, HRS could provide further insight into the intricacies of regular translation and recoding deviations
such as Programmed Ribosomal Frame shifting (PRF). Further investigations would aid in the conquest to find
new drug targets, and thus more effective antibiotics and antivirals. Emerging resistant bacteria as Methicillinresistant Staphylococcus aureus (MRSA) has proven to be a serious hazard with a wide pathological and
economical impact on the health-sector and general population.2, 3. We aim to investigate this novel state and its
possible involvement in the intrinsic helicase activity of the ribosome, and frameshifting using single molecule
Forster Resonance Energy Transfer (smFRET). In addition, we are developing a bicistronic fluorescence
construct to quantify PRF with future applications in drug screening and evolutionary studies.
Poster I-23: Correlation of the L1 Stalk Motion and Subunit Rotation
Drew E. Menke and Peter Cornish
University of Missouri
Examination of the dynamic motions of the ribosome during translation elongation has provided critical insight
into the mechanism of protein synthesis. Previously, a number of studies have examined these key dynamic
motions and have provided a detailed analysis of both the motions of the L1 stalk and inter-subunit rotation. The
L1 stalk an rRNA/protein protrusion positioned by the Exit site on the large subunit populates three states: open,
half close and closed. Subunit rotation the counter clockwise rotation of the large and small subunits opposite one
another populated the classical (nonrotated) and hybrid (rotated) states. Current results cannot directly correlate
the contribution of these two dynamic motions together. Using single molecule Fluorescence Resonance Energy
Transfer (smFRET) and a hybrid constructs labeled with three Fluorescent dyes we will examine both subunit
rotation and the L1 stalk simultaneously.
Poster I-24: Single-molecule FRET Study of the Dpo4 Behavior at DNA Containing
Benzo[a]pyrene Adduct
Pramodha S. Liyanage, David Rueda and Louis Romano
Wayne State University
Benzo[a]pyrene (BP) is a polyaromatic hydrocarbon (PAH) which can be metabolically activated to highly
reactive benzo[a]pyrene diol epoxides (BPDE) in mammals. BPDE readily reacts with DNA generating N2-dG
covalent adducts. These BP adducts are known to be extremely carcinogenic and tumorigenic in animal models.
Normally, high fidelity polymerases are unable to synthesize DNA across the BP lesions, and get stalled at the
adduct position. Translesion synthesis (TLS) is the process by which bulky adducts and other DNA damage are
bypassed, rescuing the stalled replisome. Y-family DNA polymerases play an important role in TLS. In this study
we use the Y-family DNA polymerase Dpo4 as a model system to investigate the bypass mechanism of a BP
adduct. We characterized Dpo4 binding to damaged DNA and subsequent conformational rearrangements by
single molecule fluorescence resonance energy transfer (smFRET).
30
Poster I-25: The Spliceosome Transiently Undocks the Splicing Substrate to Promote
Catalysis, Proofreading, and Alternative Splicing
Mario Blanco, Daniel Semlow, Jonathan Staley, Nils Walter and Yi Zeng
University of Chicago
Gene expression requires high fidelity at all stages. In contrast to fidelity mechanisms in transcription and
translation, fidelity mechanisms in splicing remain poorly understood. To ensure fidelity, the spliceosome
employs DExD/H-box ATPases to discriminate against suboptimal splice sites, but it has remained unclear how
these factors promote intron excision with high specificity. By assaying substrate conformation by single
molecule fluorescence energy resonance transfer, we revealed that the DExD/H-box ATPases Prp16 and Prp22
proofread and promote splice site selection at the catalytic stage through a common mechanism of substrate
undocking. Although Prp16 is canonically required only for exon ligation, we unexpectedly discovered that in the
Prp16-dependent rejection of suboptimal branch sites, undocking allowed re-docking and selection of alternative
branch sites, establishing that spliceosomal DEAH-box ATPases can function as RNA chaperones to facilitate
alternative splicing. Our observation that Prp16 promotes alternative branch site selection also permitted us to
investigate the mechanism of Prp16 function in a manner that is uncoupled from 3’ splice site recognition and
exon ligation and readily dissect the substrate requirements for Prp16-dependent branch site undocking. Our
preliminary results indicate that Prp16, like the spliceosomal DExD/H-box ATPases Prp2 and Prp22, acts on
single stranded substrate RNA downstream of the catalytic core. These data strongly suggest a common
mechanism for DExD/H-box ATPases at the catalytic stage involving 3’ to 5’ translocation along the splicing
substrate to transiently disrupt the catalytic core and facilitate splice site selection. The function of spliceosomal
DEAH-box ATPases at the catalytic stage can therefore be likened to the resolution of non-native folding
intermediates that impede activation of a catalytic ribozyme.
Poster I-26: Intramolecular Head-to-tail Distance in 2D Reveals That Kinesin Walks With Its
Cargo Upright
Kai Wen Teng, Marco Tjioe, Carol S. Bookwalter, Kathleen M. Trybus and Paul R. Selvin
University of Illinois at Urbana-Champaign
In the cell, kinesin works with different motors in order to carry various cargoes ranging from proteins to cellular
organelles towards the plus end of the microtubule. Cargo shared by kinesin, other motors, and accessory proteins
has to be carefully positioned due to steric factor. The question we wish to answer is: On a single molecule level,
how does kinesin carry its cargo across the microtubule? A glance at the structure of kinesin shows that
immediately upsteam from the motor domain is the neck coiled-coil region, which was found to orient tangential
to the microtubule in previous studies. Do the stalk and the cargo binding region of kinesin continuously extend
from the neck coiled-coil region? Or do flexible elements (hinges) play a role in positioning the cargo? Through
the use of single molecule high resolution colocalization (SHREC) we track the position of the cargo and motor
domain as kinesin is actively walking under low ATP concentration. What we found was that the cargo is not
oriented tangential to the microtubule, but it sits mostly above the motor domain. Distance between the cargo and
motor domain inferred that majority of the stalk sits upright in the z-axis. We also found that kinesin has the
freedom to position its cargo either in front or in the back of its motor domain, resulting in a wider distribution of
cargo position in the front and back, versus sideways.
31
Poster I-27: Characterization of Kinesin-1 Motor Domain Residues Important for Localization
within Neurons
Michael Kelliher, Aaron Hoskins and Jill Wildonger
University of Wisconsin-Madison
Work with kinesin in cultured neurons showed that a constitutively active kinesin-1 construct (K560) is capable
of localizing to the distal tips of developing neurites that ultimately become axons. Other work has implicated
residues within the β5-L8 and α4-L12-α5 regions of the kinesin-1 motor as being important for proper
localization. In this work we introduce the mutations made to the K560 constructs into the Drosophila
melanogaster kinesin-1 and characterize the effects on neuronal morphology and polarized transport within
neurons in live fruit flies. We are also carrying out companion single-molecule motility assays on the K560
mutants to gain insight into how these mutations affect the behavior of individual motors.
POSTER SESSION II
Poster II-1: Folding Dynamics of a WW Domain Protein Using Single Molecule Force
Spectroscopy
Miles L. Whitmore, Dena Izadi, Lisa J. Lapidus and Matthew J. Comstock
Michigan State University
We present high-resolution force spectroscopy measurements of the protein folding dynamics of a single WW
domain. Protein folding, the process by which the polypeptide chains acquire the correct three-dimensional
structure is still a poorly understood process. Force is a natural technique for denaturing proteins in order to
observe the process of refolding in detail, but the time resolution of standard instruments typically preclude
investigation of fast-folding model proteins. In this study we added cysteine residues to both N and C termini of
the human Yes-associated protein (hYAP), which typically folds in less than 1 ms. The peptide is then connected
to two 1.5 kb thiol-modified double stranded DNA handles. The protein-DNA chimeras were suspended between
a pair of polystyrene beads held in high-resolution dual optical traps. Reversible protein folding and unfolding
was observed both during force-extension pulling/relaxation experiments as well as under constant force feedback
conditions. We also labeled the N and C termini of the WW domain protein with donor and acceptor fluorophores
and performed simultaneous and independent FRET measurements of folding and unfolding using combined
high-resolution trapping and single molecule fluorescence spectroscopy.
32
Poster II-2: Resolving Prion Protein Aggregation at the Single Molecule Level
Chi-Fu Yen, Anumantha Kanthasamy and Sanjeevi Sivasankar
Iowa State University
Transmissible Spongiform Encephalopathies (TSEs) are a class of neurodegenerative disorders characterized by
the accumulation of misfolded prion protein (PrP) aggregates in the brain. A key step in this aggregation process
is the conversion of proteinase-K (PK) sensitive PrP (PrPsen) into a pathological isoform that resists PK digestion
(PrPres). Metal ions are known to play an important role in promoting PrP misfolding and oligomerization;
however, the underlying mechanisms are poorly understood at molecular level. To address these questions, we
developed single molecule assays to monitor the conversion of PrPsen into PrPres and to compare their
interaction kinetics. Using single molecule fluorescence based PK-resistance assay, we demonstrate that PrPsen
monomers convert to a PrPres conformation before oligomer assembly. The unstructured N-terminal region of
PrPsen and Cu2+ ions are essential cofactors for structural transition. Using single molecule force measurements
with an Atomic Force Microscope (AFM), we show that the association rate between monomeric PrP is reduced
upon removing the N-terminal region. Compared to the PrPsen isoform, PrPres monomers show a 900 fold higher
affinity (KA), indicating their potential as seeds for subsequent formation of prion protein aggregates.
Poster II-3: Resolving the Molecular Mechanism of Cadherin Catch Bond Formation
Kristine Manibog, Hui Li, Sabyasachi Rakshith and Sanjeevi Sivasankar
Iowa State University
Classical cadherin Ca2+-dependent cell-cell adhesion proteins play key roles in embryogenesis and in
maintaining tissue integrity. Cadherins mediate robust adhesion by binding in multiple conformations. One of
these adhesive states, called an X-dimer, forms catch bonds that strengthen and become longer lived in the
presence of mechanical force. Here, we use single molecule force clamp spectroscopy with an Atomic Force
Microscope along with Molecular Dynamics and Steered Molecular Dynamics simulations to resolve the
molecular mechanisms underlying catch bond formation and the role of Ca2+ ions in this process. Our data
suggest that tensile force bends the cadherin extracellular region such that they form long-lived, force induced
hydrogen bonds that lock X-dimers into tighter contact. When Ca2+ concentration is decreased, fewer of these
hydrogen bonds are formed and catch bond formation is eliminated.
Poster II-4: Resolving the Molecular Mechanism of Cadherin Ideal Bond Formation
Sunae Kim, Kristine Manibog, Sabyasachi Rakshith and Sanjeevi Sivasankar
Iowa State University
Classical cadherin cell-cell adhesion proteins play an essential role in maintaining tissue integrity in the presence
of mechanical stress. Cadherins bind in multiple adhesive conformations; by switching between these
conformations, cadherins control the mechanical properties of their interactions. We had previously shown that
while one cadherin conformation forms ‘catch’ bonds which strengthen with force, the second conformation
forms ‘slip’ bonds which weaken when pulled. We had also determined that cadherins form a third, previously
unknown, type of interaction called an ‘ideal’ bond that is insensitive to force. While we have recently resolved
the molecular mechanisms of cadherin catch and slip bond formation, the molecular determinants by which ideal
bonds form are unknown. To resolve this, we use single molecule force clamp measurements with an Atomic
Force Microscope to characterize the molecular basis of cadherin ideal bond formation.
33
Poster II-5: Characterizing the Interaction of Desmosomal Cadherins at Single Molecule Level
Omer M. Shafraz, Sabyasachi Rakshith, Molly Lowndes, W. James Nelson and Sanjeevi Sivasankar
University of Illinois at Urbana-Champaign
Desmosomes are cell-cell adhesion complexes that are present in tissues that resist mechanical stress. They are
mainly composed of two adhesive proteins which are members of the cadherin superfamily of cell adhesion
proteins, desmocollin (Dsc) and desmoglein (Dsg). However, the role of these proteins in desmosomal adhesion is
unclear. Here, we use the single molecule force spectroscopy with an Atomic Force Microscope (AFM-FS) to
characterize the interactions of type-2 isoforms of desmocollin (Dsc2) and desmoglein (Dsg2). We show that
Dsc2 forms Ca2+ dependent homophilic bonds by swapping a conserved Tryptophan residue between opposing
binding partners; mutating this Trp inhibits Ca2+ dependent homophilic binding. In contrast, Dsg2 forms Ca2+
independent heterophilic bonds with Dsc2 via a mechanism that does not involve Trp strand-swapping.
Previous studies suggest that desmosome formation requires the presence of classical cadherins at the site of
desmosome assembly. This suggests a cross-talk between desmosomal and classical cadherins at cell-adhesion
contacts. We therefore used AFM-FS to test if Dsc2 and Dsg2 interact with E-cadherin, a classical cadherin
present in the epithelium. Our data shows that while Dsc2 does not bind to E-cadherin in the presence of Ca2+,
Dsg2 forms Ca2+ independent complexes with E-cadherin. Using cadherin mutants we show that the interactions
between Dsg2 and E-cadherin occur via a previously uncharacterized binding interface that does not involve
either Trp strand-swapping or X-dimer formation (two well established classical cadherin binding mechanisms).
Poster II-6: Multiple Conformations of a Single SNAREpin between Two Nanodisc
Membranes Reveal Diverse Pre-fusion States
Jaeil Shin, Xiaochu Lou, Dae-Hyuk Kweon and Yeon-Kyun Shin
Iowa State University
SNAREpins must be formed between two membranes to allow vesicle fusion, a required process for
neurotransmitter release. Although its post-fusion structure has been well characterized pre-fusion conformations
have been elusive. We used single molecule FRET and EPR to investigate the SNAREpin assembled between
two nanodisc membranes. The SNAREpin shows at least three distinct dynamic states, which might represent
pre-fusion intermediates. While the N-terminal half above the conserved ionic layer maintains a robust helical
bundle structure the membrane-proximal C-terminal half shows either high FRET representing a helical bundle
(45%), low FRET reflecting a frayed conformation (39%), or mid FRET revealing an yet unidentified structure
(16%). It is generally thought that SNAREpins are trapped at a partially zipped conformation in the pre-fusion
state, and complete SNARE assembly happens concomitantly with membrane fusion. However, our results show
that the complete SNARE complex can be formed without membrane fusion, which suggests that the complete
SNAREpin formation could precede membrane fusion, providing an ideal access to the fusion regulators such as
complexins and synaptotagmin 1.
34
Poster II-7: Dynamics of the General Secretory System Viewed in Near-native Conditions via
Atomic Force Microscopy
Raghavendar Reddy Sanaganna Gari, N.C. Frey, L.L. Randall and Gavin M. King
University of Missouri
In bacteria and archaea the protein conducting channel SecYEG provides a ubiquitous pathway for protein
transfer across and into membranes. Further, it is known that SecA is the ATPase of the general secretory system
and it binds SecYEG to perform translocation. In so doing, SecA makes large surface area contact with the
unstructured cytoplasmic loops spanning transmembrane helices 6-7 and 8-9 of SecY. Despite their functional
significance, measurements of flexible and disordered protein domains remain a significant experimental
challenge. Recently, atomic force microscopy (AFM) has emerged as an important complementary tool in
biophysics and is well suited for studying membrane protein dynamics in near-native conditions (i.e., in a native
lipid environment, at physiologically relevant temperature and ionic strength). We have studied purified SecYEG
that was reconstituted into liposomes via AFM. After confirming activity, changes in the structure of SecYEG as
a function of time were directly visualized. The dynamics observed were significant in magnitude and were
attributed to the aforementioned loops of SecY. In addition, we identified a distribution between monomers and
dimers of SecYEG as well as a smaller population of higher order oligomers. Finally, we have imaged SecA
engaged on SecYEG and related the structural states observed to the activity of the translocase. This work
provides a novel and near-native vista of central components of the general secretory system.
Poster II-8: The Synaptotagmin 1 Linker May Function as an Electrostatic Zipper That Opens
for Docking but Closes for Fusion Pore Opening
Xiaochu Lou, Ying Lai, Yongseok Jho, Tae-Young Yoon and Yeon-Kyun Shin
Iowa State University
Synaptotagmin 1 (Syt1), a major Ca2+ sensor for fast neurotransmitter release, contains tandem Ca2+-binding C2
domains (C2AB), a single transmembrane a-helix, and a highly charged 60-residue-long linker in between. Using
the single vesicle docking and content mixing assay we found that the linker region of Syt1 is essential for its two
signature functions: Ca2+-independent vesicle docking and Ca2+-dependent fusion pore opening. The linker
contains the basic amino acid-rich N-terminal region and the acidic amino acid-rich C-terminal region. When the
charge segregation was disrupted, fusion pore opening was slowed while docking was unchanged. Intramolecular
disulfide cross-linking between N- and C-terminal regions of the linker or deletion of 40 residues from the linker
reduced docking while enhancing pore opening. The EPR analysis showed Ca2+-induced line broadening
reflecting a conformational change in the linker region. Thus, the results suggest that the electrostatically bipolar
linker region might have the capacity to extend for docking and fold to facilitate pore opening.
35
Poster II-9: Real Time Observation of Lipid-Protein Interactions in Crude Cell Lysates and
with Single-Molecule Resolution
Edwin Arauz, Vasudha Aggarwal, Ankur Jain, Taekjip Ha, and Jie Chen
University of Illinois at Urbana-Champaign
Lipid-protein interactions play key roles in signal transduction. Obtaining new mechanistic insights of these
interactions is obligatory for a better understanding of biological processes. Here we use a single-molecule pulldown assay (SiMPull) to probe lipid-protein interactions in crude cell lysates. We demonstrate the applicability of
this assay by showing specific interaction between several signaling lipids and their lipid-binding partners. We
perform intensive single-molecule data analysis to quantitatively describe the assembly lipid-binding proteins on
their target lipids. Importantly, this assay is applicable to full-length proteins expressed in crude cell lysates, as
show for the protein kinase AKT which binds to PI(3,4,5)3 lipid specifically. This new assay lays the foundation
to study the interaction of large macromolecular complexes with lipids second messengers in cell lysates,
avoiding the need of harsh and lengthy procedures used during protein purification.
Poster II-10: Single-molecule Studies of Membrane Proteins on Glass Substrates Using
Atomic Force Microscopy
Nagaraju Chada, Krishna P. Sigdel, Tina R. Matin, Raghavendar Reddy Sanganna Gari, Chunfeng Mao, Linda
L. Randall, and Gavin M. King
University of Missouri
Since its invention in the mid-1980s, the atomic force microscope (AFM) has become a valuable complementary
tool for studying membrane proteins in near-native environments. Historically, mica is the most common
substrate utilized for biological AFM. Glass being amorphous, transparent, and optically homogeneous has its
own set of advantages over mica and has the potential to broaden the overlap of AFM with techniques that require
high quality non-birefringent optical access. The use of silanized glass as an AFM substrates has been reported as
a means to fine tune surface chemistry. However, such coatings usually require hours of additional preparation
time and can lead to increased surface roughness. In this work, we present a simple technique for preparing
borosilicate glass as a substrate for two membrane protein systems: non-crystalline translocons (SecYEG) of the
general secretary system from E. coli, and bacteriorhodopsin (BR) from H. salinarum. For both these membrane
proteins, quantitative comparisons of the measured protein structures on glass versus mica substrates show
agreement. An additional advantage of glass is that lipid coverage is rapid (< 1 minute) and complete (occupying
the entire surface). A goal is to study the bacterial export system using recently developed precision measurement
techniques such as ultra-stable AFM.
36
Poster II-11: Cellular Pathways for Productive HIV-1 Entry and Molecular Mechanisms of its
Inhibition
Hanna Song and Wei Cheng
University of Michigan
Productive entry of the human immunodeficiency virus type I (HIV-1) into CD4+ T cells is initiated by binding
of the viral envelope gp120 to CD4 receptor. This binding causes a cascade of conformational changes in both the
gp120 and gp41 that eventually lead to viral-cell membrane fusion and HIV-1 entry. Early studies have suggested
that HIV-1 can enter target cells via direct fusion at the plasma membrane. In contrast, recent studies have
suggested that the direct fusion at the plasma membrane is not productive. Instead, HIV-1 may enter cells via
dynamin-dependent endocytosis. To examine the extent to which endocytosis leads to productive infection of
HIV-1, we have used several inhibitors of dynamin to investigate whether there is a correlation between
inhibition of HIV-1 infection and the inhibition of cell endocytosis. Transfection of TZM-bl indicator cells by
dyn1(K44A), the dominant-negative mutant of dynamin I, decreased HIV-1 infection by ~30%, which correlated
with the decrease of endocytosis as monitored via transferrin uptake, suggesting that dynamin-dependent
endocytosis contributes to the productive infection of HIV-1. Dynasore, a noncompetitive inhibitor of dynamin,
inhibited HIV-1 infection in various cell lines, but this inhibition is not correlated with the reduction in transferrin
uptake, suggesting that dynasore inhibits HIV-1 infection through an off-target effect. T20, the membraneimpermeable inhibitor of HIV-1 entry, potently inhibited HIV infection in all cell lines investigated.
Interestingly, endocytosed HIV-1 can be clearly observed in host cells even in the presence of saturating T20,
suggesting that T20 may be endocytosed together with HIV-1 and exert its effect inside an endosome. This
suggestion is further supported by the fact that inhibition of viral endocytosis doesn’t change the efficacy of T20.
Ongoing colocalization studies of HIV-1, endosomal marker, and fluorescent-labeled T20 at single-molecule
resolution will definitely test this hypothesis (Supported by March of Dimes Foundation, 5-FY10-490; WC).
Poster II-12: Cell Geometry Affects the Distribution of Measured Diffusion Coefficients in
Bacteria
David J. Rowland and Julie S. Biteen
University of Michigan
Fast diffusion in small volumes is a phenomena important to many biophysical studies dealing with single
molecules confined to micron-sized volumes such as membrane domains or bacteria. Presented here is the
simulated transition from free to confined diffusion in a half-micron diameter bacterial cell as measured by mean
squared displacement (MSD) analysis. Partially confined motion in an asymmetric volume can separate a single
diffusing population into two, prompting the use of a two-population diffusion model. The use of doubleexponential fits to cumulative step size probability curves underestimates the diffusion coefficient, however, and
we show that geometrically separating the diffusion into axial and transverse directions more precisely predicts
the real diffusion coefficient. Two orthogonal methods of calculating the MSD curve are used. We compare the
traditional single-molecule super-resolution technique of fitting particle point spread functions with
spatiotemporal image cross correlation spectroscopy (STICS) which does not suffer from particle blurring as a
result of long integration times or fast diffusion. Both show that geometric separation of step sizes more precisely
predicts the true diffusion coefficient than does a two-term fit to the whole data set and STICS more accurately
measures fast diffusion.
37
Poster II-13: Spatiotemporal Dissection of Cytoplasmic and Nuclear miRNA Function
Laurie Heinicke, Sethuramasundaram Pichiaya, Elizabeth Cameron and Nils Walter
University of Michigan
Endogenous microRNA (miRNA) genes are transcribed in the nucleus as primary miRNA transcripts (primiRNA) and processed via multiple steps to generate mature miRNAs in the cytoplasm. These small non-coding
RNA (ncRNAs) associate with components of the RNA-induced silencing complex (RISC) and engage mRNA
targets and regulate gene repression via translational inhibition and/or mRNA degradation. Despite rapid
advances in our understanding of miRNA biogenesis and mechanism, the intracellular dynamics and assembly of
miRNA-associated complexes and the spatiotemporal modulation of miRNA-regulated gene expression are still
unclear. To uniquely probe intracellular RNA silencing pathways, our lab has developed a method termed
intracellular Single-molecule High-Resolution Localization and Counting (iSHiRLoC) to determine the
localization, diffusion constant and assembly state of single miRNA complexes inside living human cells at 30
nm spatial and 100 ms temporal resolution. We have used iSHiRLoC to probe nuclear functions of miRNAs. We
found that a significant fraction of microinjected mature let-7-a1 (~20-30%) localizes to the nucleus, in contrast to
a control cxcr4 miRNA whose whereas nuclear localization was minimal (~5-10%). Inhibition of transcription
reduced nuclear localization of let-7-a1 to ~5-10%, the level of cxcr4 in the absence or presence of transcription
inhibitor, strongly suggesting that only let-7-a1 binds RNA targets in the nucleus. These observations are
consistent with a recent report that describes autoregulation of let-7-a1 biogenesis in the nucleus by mature let-7a1. We are currently pursuing time course experiments, subcellular fractionation followed by Northern blotting
and knock down of miRNA-associated proteins to better understand the import mechanism and target engagement
of nuclear miRNAs. Together, these studies highlight the versatility of iSHiRLoC by providing biological insight
regarding assembly and localization of intracellular miRNAs.
Poster II-14: Single-molecule Fluorescence Imaging of RecO Localization and Dynamics in
Bacillus subtilis
Hannah H. Tuson, Yi Liao, Lyle A. Simmons and Julie S. Biteen
University of Michigan
In all organisms, the high fidelity of DNA replication is essential for maintenance of chromosome integrity. DNA
damage can be caused by polymerase errors or by external factors (e.g., X-rays or mutagenic chemicals). Thus,
the cell has evolved a number of repair mechanisms to respond to different types of damage. In B. subtilis, repair
of double-strand breaks (DSBs) in the DNA occurs through RecA-mediated homologous recombination. This role
for RecA in DSB repair in B. subtilis is analogous to that of Rad51 in eukaryotes, making B. subtilis an excellent
model system for studying cellular response to DNA damage. The mechanism by which RecA finds DSBs in vivo
is not well described, but is believed to involve the proteins RecF, RecO, and RecR. Previously, bulk fluorescence
studies have shown that RecO forms foci after the induction of double-strand breaks. However, RecO in
undamaged cells can only be visualized when over-expressed, leaving questions about its true localization at wild
type expression levels. Here, we have created cells in which PAmCherry-RecO is natively expressed from the
RecO promoter as the only RecO source. We use single-molecule fluorescence microscopy in live B. subtilis to
show that RecO is generally diffuse throughout the cell. This result suggests that, unlike several other proteins
involved in DNA repair, RecO is not associated with the replisome prior to DSB recognition. Furthermore, RecO
dynamics change when cells are treated with the DNA-damaging agent phleomycin. Additionally, we are
examining the co-localization of RecO with SSB (which recruits RecO) and RecA (which is recruited by RecO).
Understanding the real-time dynamics, positioning, and interactions of RecO will yield a model for the early
stages of DSB recognition and repair and further inform our general understanding of DNA damage repair.
38
Poster II-15: Single-molecule Mechano-memory
Isaac T.S. Li, Taekjip Ha, and Yann R. Chemla
University of Illinois at Urbana-Champaign
Understanding the spatial distribution of individual adhesion bonds and the tension exerted on them is crucial for
understanding whole cell adhesion behavior. Here, we introduce a new class of molecular force sensors to record
cellular adhesion events at the single-molecule level. A DNA structure was designed that responds to mechanical
perturbation above certain threshold tension and maintains a memory of that perturbation. We name this feature
“single-molecule mechano-memory” (smMM). The smMM sensor undergoes conformational changes under
tension and is kinetically trapped under a new conformation. Single-molecule force spectroscopy and
fluorescence spectroscopy were performed to characterize the activation force as well as memory life time. We
show that in the absence of mechanical perturbation the smMM sensor is well folded and stable. In the presence
of tension above ~35 pN, the sensor is converted to the unfolded “memory” state in which it remains kinetically
trapped for an average of 25 seconds. Both activation force and life time of the sensor can be tuned by its DNA
sequence. As a proof of concept for this class of sensors, smMM sensors were coated on a surface where cell
adhesion takes place. Individual adhesion events are detected using fluorescently-labeled oligonucleotide probes
to mark unfolded sensors.
Poster II-16: Single-molecule Tracking of Elongation Factor P in Live E. coli Cells
Heejun Choi, Sonisilpa Mahapatra, Suparna Sanyal and James C. Weisshaar
University of Wisconsin-Madison
Elongation Factor P (EF-P) is essential for efficient peptidyl transfer for cellular growth by enhancing the rate of
polyproline addition in peptide elongation. In vitro studies showed that EF-P is associated with ribosome at its
exit site. We are interersted in understanding the ribosome-EF-P interaction in live cells. Here, we constructed a
translational fusion of mEos2 appended to C-terminus of efp in chromosome. Using single molecule tracking
PALM and superresolution imaging, we are able to study the subcellular diffusion and distribution of EF-P in live
cells. Tracking of single molecules of EF-P-mEos2 showed that EF-P diffuses at 1.4 µm2/s and showed signs of
ribosome-association. Implication of translation and transcription halting drugs on association of EF-P to
ribosome will be discussed.
39
Poster II-17: The Princess and the Pea: A Story of Cell Mechanics
Mehdi Roeimpeikar, Qian Xu and Taekjip Ha
University of Illinois at Urbana-Champaign
Single molecules of integrins, a class of trans-membrane proteins involved in cell adhesion, pull on their ligands
with a force of approximately 40 pN. Measurements of these forces were previously performed using a series of
double stranded (ds) DNA, each with a different rupture force, conjugated to the ligand. DNA types with rupture
forces below 40pN are easily pulled apart by cells (through engagement with integrins). Above 40 pN, cells
remain attached. In this research, using this series of different types of DNA tethers, two-dimensional force
spectroscopy can be performed through multiplexing two different types of DNA tethers.
Poster II-18: Somitogenesis in Zebrafish
Zhengda Li, Ye Guan and Qiong Yang
University of Michigan
Biological clock is a vital component of organisms, which determines the specific timing of biological event
sequences. During embryogenesis, robust time tuning not only helps coordinated differentiation, but also leads to
accurate morphogenesis. Understanding the interplay of clocks in developments will not only help investigation
of fundamental questions like organism size control, pattern formation or cell fate determination, but also cast
light on smarter medicine usage schemes or better drugs dealing with abnormally differentiated cells. However, at
present, even though under intense investigation, the specific role of biological clocks in development is still far
from clear.
Our research is to investigate the segmentation process of zebrafish. Somitogenesis is a common phenomenon for
nearly all vertebrates. Segments, also called somites, form early and develop to skeleton muscle, epithelial cells
and many other structures. The study of somite formation may trace back to 1970s when many possible
mechanisms are proposed. Further research unveiled a conserved molecular mechanism among vertebrates, and
presented a model: clock and wavefront model, which prevailed for decades. However, with the development of
microscopy and molecular biology, more evidence are emerging which proposed new functional components or
even challenged the classical model. We build preliminary model based on current information, and find that to
explore the interplay of cell cycles and segmentation clocks would be of great interests. Using in toto imaging, we
can track the phase of different oscillators as well as the motion and proliferation of each cell, and our final goal
is to understand the how global regulation and local interaction works together to produce stable patterns in
organisms, which would rely on the further development of the single molecular imaging. Our research will help
to understand the mechanism of somitogenesis and to expand our knowledge on coupled oscillators in biological
systems.
40
Poster II-19: Virion Immobilization Post Diffusion Limits HIV-1 Infectivity Revealed by
Real-time Single Particle Tracking
Michael C. DeSantis, Jin H. Kim, Jamie L. Austin, and Wei Cheng
University of Michigan
HIV-1 is known to have low infectivity. Although virion-cell interactions that take place prior to viral entry have
been suggested to limit the infectivity of the virus, the underlying mechanisms are not completely understood.
Furthermore, receptor binding, in addition to nonspecific interactions at the cell surface, might influence the
subsequent steps, i.e., entry pathways, of infection. Using single-particle tracking methods with spatial and
temporal resolutions of ~20 nm and 40 ms, respectively, we have quantified the dynamics of mCherry labeled
HIV-1 virions with varied envelope (Env) glycoprotein incorporation interacting with TZM-bl cells, a HeLaderived cell line that is commonly used for quantitation of HIV-1 infection. Our analysis revealed that the
frequency of viral-cell encounters is consistent with diffusion-limited interactions. However, most virions only
have transient interactions with the cell surface before permanent dissociation. As a result, the probability for a
single virion to become immobilized upon encounter with a cell is at least an order of magnitude lower than the
frequency of initial collision. The majority of these immobilized virions enter cells via endocytosis with
efficiencies as high as 75%. However, highly efficient endocytic uptake is independent of either viral Env or
coreceptors on target cells such that it can effectively reduce the pool of infectious virions. DEAE-dextran, a
reagent known to enhance viral titers, dramatically increased the rate of immobilization in a manner independent
of viral Env content. Moreover, the presence of DEAE-dextran decreased endocytic uptake by twofold which
may afford virions greater opportunity to bind specifically with receptors thereby increasing HIV-1 infectivity. In
summary, these studies suggest that the low probability for immobilization upon target cell encounter as one
potential bottleneck to HIV-1 infectivity. Reagents that can increase immobilization or decrease coreceptorindependent endocytosis can dramatically enhance HIV-1 infectivity. (Supported by NIH DP2-OD008693 and
F32-GM109771)
Poster II-20: Electrical Current Measurement and Manipulation of Single Geobacter Cells Via
Optical Trapping
Jess L. West, Adam J Gros, Rebecca J Steidl, Gemma Reguera and Matthew J Comstock
Michigan State University
The ability of Geobacter bacteria to respire to extracellular electron acceptors such as uranium and electrodes
shows promise for applications in bioremediation and bioenergy. Key to these technologies is a better
understanding of how individual cells transfer electrons outside the cell to extracellular electron acceptors. Bulk
measurements of cells attached to microfabricated electrodes indicate that a single cell may be able to generate
current ranging from 100 to 1,000 fA. We have constructed an experimental setup integrating optical tweezers
and sample chambers equipped with a micron-scale gold electrode that allow in situ current measurements down
to 50 fA. We have trapped individual Geobacter sulfurreducens cells and we can precisely manipulate them onto
a gold electrode while simultaneously measuring current. With this technique, we are interrogating the cellular
components responsible for current production in Geobacter.
41
Poster II-21: Optical Trapping and Characterization of Single HIV-1 in Culture Media
Yuanjie Pang and Wei Cheng
University of Michigan
Optical trapping uses the momentum change of photons to impart forces on microscopic objects, thereby
immobilizing and manipulating these objects in three dimensions. Optical trapping may have great potential in
microbiology applications because of its ability to trap tiny biological particles in solution. Here we demonstrate
trapping of single HIV-1 virions in culture media using the single-beam gradient optical tweezers, and
simultaneous multi-parameter characterizations of the trapped virion based on its thermal motion recorded by an
ultra-sensitive position sensing diode (PSD) at the back-focal-plane (BFP) of the optical tweezers. By fitting the
virion’s thermal motion power spectrum to a Lorentzian function; we obtained two important parameters, Dvolt, a
proportionality factor, and fc, the corner frequency, of the Lorentzian. Both parameters conform to different
statistical distributions for single virions as opposed to apparent aggregates. We then calibrated the electronic
signal from the PSD using spatial distance values by sinusoidally oscillating the sample chamber at a known
amplitude and frequency. This calibration allowed us to convert Dvolt into the hydrodynamic diffusion
coefficient, and to subsequently calculate the size and the trap stiffness of the virion. The size information further
revealed the aggregation status of the virions in the culture media that was hidden behind Dvolt and fc
distributions, and virion aggregation was found to be concentration-dependent. Furthermore, by fitting the sizestiffness relation to a theoretical model, we were able to deduce the refractive index of the virion, which is an
important parameter for modelling optical biosensors. We believe the single virion manipulation technique will
open a new path in virology studies. For example, a single virion to a single cell infection assay can be set up,
which can potentially reveal the heterogeneity of infection among individual virions from a viral population
(Supported by NIH Director’s New Innovator Award 1DP2OD008693).
Poster II-22: Combined Multi-color Fluorescence and Ultra-High Resolution Optical Tweezers
Cho-Ying Chuang, Miles L Whitmore, Jess L West, and Matthew J Comstock
Michigan State University
We present a single-molecule instrument that combines ultra-high resolution optical tweezers with multicolor
confocal fluorescence microscopy. Timeshared dual optical traps were interlaced and synchronized with three
fluorescence excitation lasers (473 nm, 532 nm, and 633 nm) and three single-photon counting detectors (one for
each excitation laser). Our new instrument enables the simultaneous measurement of DNA tether extension
changes (e.g., from helicase or polymerase motion) and multiple fluorescently labeled observables (e.g., internal
protein conformation dynamics via FRET or precise stoichiometry of complexes via multi-colored fluorophore
counting). We demonstrated our instrument by measuring the binding and unbinding of fluorophore-labeled
single stranded DNA oligonucleotides to a complementary tethered strand of DNA. Further, we combined multichannel sample chambers with precise computer control of fluorescence measurement and triggered chamber
motion to implement an automated ‘molecular assembly line.’ This allowed us to precisely add individual
molecules of different types to a single DNA tether while conserving fluorescence photons and reducing
photobleaching. In the future, these instrumentation advancements should enable the precise single-molecule
assembly and measurement of complex, multi-component molecular machine systems.
42
Poster II-23: Three Dimensional Localization of Single Biomolecules with Nanometer
Resolution
Patrick D. Schmidt, Chi Fu Yen, John Lajoie and Sanjeevi Sivasankar
Iowa State University
Super-resolution fluorescence methods are widely used to image single biomolecules with sub-diffraction
resolution. While these techniques can localize fluorophores with nm resolution in the x- and y- directions, their
resolution along the optical (z) axis is poor. To overcome this limitation, we recently developed a fluorescence
technique called Standing Wave Axial Nanometry (SWAN) that can localize single molecules along the z-axis of
a fluorescence microscope with nm accuracy and precision. Here, we present our progress in integrating SWAN
with 2D fluorescence localization methods to achieve previously unprecedented, nm localization accuracy in all
three spatial dimensions. We will focus on our recent progress in designing fast feedback loops that stabilize the
microscope over long periods of time and eliminate nm scale instrumental drift that occurs on the relevant
timescale for data acquisition. Compensating for thermal and mechanical drifts should enable us to image single
molecules in three dimensions with nm localization accuracy in each dimension.
Poster II-24: Site-specific Labeled HIV-1 Neutralization Antibodies for Single-molecule
Fluorescence Measurement
Jin H. Kim and Wei Cheng
University of Michigan
To quantify the copy number of proteins using single-molecule fluorescence, specific labeling of the protein with
a fluorophore in high efficiency is required. An antibody containing only a single antigen binding site conjugated
with a single fluorophore provides the ideal condition for correlating the copy numbers between antigens of
interest and the fluorophores on the antibody. In general schemes to achieve such a labeled antibody, sulfhydryl
groups in mildly reduced half antibody molecules were used for labeling with fluorophores via maleimide
chemistry. However, the uncertainty in preparation of such a half antibody and the nonspecific reduction of
multiple sulfhydryl groups in an antibody molecule render this scheme highly unreliable. Here, we have
developed a method to conjugate a single Alexa fluor 594 dye to an antibody containing only a single antigen
binding site. To prepare an antibody with only a single antigen binding site and a single free sulfhydryl group
available for maleimide conjugation, the heavy chain genes of two HIV-1 neutralizing antibodies, VRC01 and
PGT145, were modified by a truncation of the Fc portions and an insertion of a hexahistidine tag after the first
cysteine in the hinge region of the IgG. The modified antibody, named VRC01 Fab” and PGT145 Fab”, were
successfully expressed in the suspension culture of 293-F cells, and purified using Ni-NTA column, followed by
conjugation with Alexa fluor 594 and further purified using gel filtration column. The final labeled VRC01 Fab”
and PGT145 Fab” exhibited higher than 95% of purity and 98% of labeling efficiency. In addition, these
antibodies neutralized HIV-1 infection in cell culture assays at levels comparable to wild type IgG antibodies.
Therefore, these site-specific labeled antibodies, VRC 01 Fab” and PGT145 Fab” will provide efficient assay
tools for investigating the copy number of envelope glycoproteins on the surface of HIV-1 virions.
43
Poster II-25: Mechanical Modulation of Enzyme Activity by Dynamic DNA Tweezers
Soma Dhakal, Minghui Liu, Matthew R. Adendorff, Mark Bathe, Hao Yan and Nils G. Walter
University of Michigan
Switchable nanomachines provide a universal platform to control dynamic systems by altering distances at the
nanoscale on-demand. Recently, a tweezers-like DNA device has been used to control the activity of an
enzyme/cofactor pair juxtaposed on the two arms of the tweezers. Initial studies focused on bulk properties of the
tweezers-mediated reactions and hence lacked insight into the mechanism of enzymatic activation. Here, we used
site-specifically fluorophore-labeled DNA tweezers and monitored the arm-to-arm distance through singlemolecule fluorescence resonance energy transfer (smFRET). The tweezers are composed of DNA double-helix
arms joined by a Holliday junction ‘hinge’. A DNA strand that can cycle between hairpin and double helical
structures through a strand-displacement reaction is incorporated between the arms to control the arm-to-arm
distance. Both smFRET and AFM measurements consistently showed that these previously designed tweezers
only partial close in the “closed” state (to an arm-to-arm distance of ~6.5 nm). We improved the design by
varying the hairpin stem-length from 3 to 5 base pairs (bp). Consistent with our smFRET results, molecular
dynamics simulations showed bending and twisting motion of the arms. Further, smFRET experiments on the
isolated Holliday junction hinge suggested that the “correct” isomer II needed to close these tweezers is relatively
disfavored, rationalizing the only partial closing of the tweezers. The performance of each tweezers design was
quantitatively assessed by juxtaposing the enzyme glucose-6-phosphate dehydrogenase (G6pDH) with its
cofactor NAD+ on the tweezers arms and measuring the G6pDH activity through a coupled enzyme cascade.
Using our optimized tweezers, we were able to enhance the activity of G6pDH by up to ~9-fold. Our strategy for
improving a DNA device using feedback from single-molecule experiments may represent a general approach to
designing refined nanodevices for future applications.
44
GRADUATE & POSTDOCTORAL PARTICIPANTS
Name
Institution
Email address
Aggarwal, Vasudha
University of Illinois at Urbana-Champaign
vasuagg@gmail.com
Bernier, Morgan
The Ohio State University
welshmj@mps.ohio-state.edu
Beyer, David
University of Iowa
david-beyer@uiowa.edu
Boehm, Elizabeth
University of Michigan
eeboehm4@gmail.com
Bourg, Julia
University of Michigan
jbourg@umich.edu
Brehove, Matthew
The Ohio State University
brehove.1@osu.edu
Brenlla, Alfonso
Wayne State University
alfonso@chem.wayne.edu
Budhathoki, Jagat
Kent State University
jbudhath@kent.edu
Cameron, Elizabeth
University of Michigan
cameroel@umich.edu
Casy, Wilder
University of Missouri
wcfyc@mail.missouri.edu
Chada, Nagaraju
University of Missouri
nckt2@mail.missouri.edu
Chen, Ran
University of Iowa
ran-chen@uiowa.edu
Choi, Heejun
University of Wisconsin- Madison
hchoi22@wisc.edu
Chowdhury, Farhan
University of Illinois at Urbana-Champaign
fchowdh3@illinois.edu
Chuang, Cho-Ying
Michigan State University
chuangc2@msu.edu
Cuculis, Luke
University of Illinois
luke.cuculis@gmail.com
DeSantis, Michael
University of Michigan
mcdesant@med.umich.edu
Dhakal, Soma
University of Michigan
sdhakal@umich.edu
Gibson, Matthew
The Ohio State University
gibson.710@osu.edu
Gros, Adam
Michigan State University
grosadam@msu.edu
Hao, Linxuan
Washington University in St. Louis
haol@wustl.edu
Hejna, Miroslav
University of Illinois at Urbana-Champaign
mhejna@illinois.edu
Hudoba, Michael
The Ohio State University
MWHudoba@gmail.com
Izadi, Dena
Michigan State University
izadiden@msu.edu
Kafle, Rudra
University of Michigan
rudrak@umich.edu
Kapil, Dave
University of Illinois at Urbana-Champaign
kapildave91@gmail.com
Ke, Haixin
Washington University in St. Louis
keh@biochem.wustl.edu
Kelliher, Michael
University of Wisconsin-Madison
mtkelliher@wisc.edu
Kim, Jin H.
University of Michigan
jinhkim@umich.edu
Kim, Sunae
Iowa State University
sunaekim@iastate.edu
45
Name
Institution
Email address
Kim, Younghoon
University of Illinois at Urbana-Champaign
younghk@illinois.edu
Kruk, Katie
University of Michigan
krukk@mail.gvsu.edu
Lee, Stephen
University of Michigan
stevalee@umich.edu
Lenhart, Justin
University of Michigan
coldnjl@umich.edu
Li, Isaac
University of Illinois
isaacli@illinois.edu
Li, Wenting
University of Wisconsin-Madison
wli73@wisc.edu
Li, Zhengda
University of Michigan
lzd@umich.edu
Lin, Chang-Ting
University of Illinois at Urbana-Champaign
clin70@illinois.edu
Liyanage, Pramodha
Wayne State University
pramodha@chem.wayne.edu
Lou, Xiaochu
Iowa State University
xlou@iastate.edu
Luo, Yi
The Ohio State University
yiluo2010@gmail.com
Manibog, Kristine
Iowa State University
kmanibog@iastate.edu
Marsh, Brenden
University of Missouri
bpmt3c@mail.missouri.edu
Menke, Drew
University of Missouri
dem4p2@mail.missouri.edu
Mohapatra, Sonisilpa
University of Wisconsin-Madison
silpasoni1@gmail.com
Ngy, Thuy
University of Illinois at Urbana-Illinois
thuyngo1@illinois.edu
Nguyen, Benh
Washington University in St. Louis
bnguyen@biochem.wustl.edu
Norman, Zenia
University of Missouri
znmq7@mail.missouri.edu
Numez, Marcos
University of Michigan
mfnunez@umich.edu
Ordabayev, Yerdos
Washington University in St. Louis
ordabayev@wustl.edu
Pang, Yuanjie
University of Michigan
yuanjiep@umich.edu
Park, Seongin
University of Illinois
jerpark@illinois.edu
Patrick, Eric
Michigan State University
patri137@msu.edu
Pennella, Min
University of Missouri
pennellami@missouri.edu
Qiu, Yupeng
University of Illinois at Urbana- Champaign
yqiu2@illinois.edu
Radzinski, Nikolai
University of Wisconsin-Madison
nick.radzinski@gmail.com
Ramreddy, Tippana
University of Illinois at Urbana-Champaign
rtippana@illinois.edu
Regan, Emma
University of Michigan
eregan2@wellesley.edu
Rodgers, Margaret
University of Wisconsin-Madison
mrodgers3@wisc.edu
Roeinpeikar, Mehdi
University of Illinois at Urbana-Champaign
roeinpe2@illinois.edu
Rowland, David
University of Michigan
djrow@umich.edu
Rube, Tomas
University of Illinois at Urbana-Champaign
rube@illinois.edu
46
Name
Institution
Email address
Sanaganna Gari, R.R.
University of Missouri
rsxf7@mail.missouri.edu
Savage, Alan
Ohio State University
savage.161@osu.edu
Schimert, Kristin
University of Michigan
schimert@umich.edu
Schmidt, Patrick
Iowa State University
pds@iastate.edu
Shafraz, Omer
Iowa State University
shafraz@iastate.edu
Shebl, Bassem
University of Missouri
bmgmw7@mail.missouri.edu
Sherani, Aiman
University of Michigan
asherani@wellesley.edu
Shin, Jaeil
Iowa State University
baldguy@iastate.edu
Sigdel, Krishna
University of Missouri
sigdelk@missouri.edu
Simenson, Angelynn
University of Missouri
aps455@mail.missouri.edu
Skootsky, Joshua
University of Michigan
joshua.skootsky@gmail.com
Sokoloski. Joshua
Washington University in St. Louis
sokoloskij@biochem.wustl.edu
Stekas, Barbara
University of Illinois at Urbana-Champaign
stekas2@illinois.edu
Su, Xin
University of Michigan
xinsu@umich.edu
Teeling-Smith, Richelle
The Ohio State University
teeling.richelle@gmail.com
Teng, Kai wen
University of Illinois at Urbana-Champaign
teng5@illinois.edu
Tomko, Eric
Washington University in St. Louis
tomkoe@biochem.wustl.edu
Tuscon, Hannah
University of Michigan
htuson@umich.edu
Wang, Jiarui
University of Michigan
jerrywon@umich.edu
West, Jess
Michigan State University
westjes3@msu.edu
Whitley, Kevin
University of Illinois in Urbana-Champaign
kwhitle2@illinois.edu
Whitmore, Miles
Michigan State University
whitmo46@msu.edu
Widom, Julia
University of Michigan
jwidom@umich.edu
Wu, Colin
University of Iowa
colin-wu@uiowa.edu
Wurm, Sarah
The Ohio State University
wurm.35@osu.edu
Yang, Zhilin
University of Wisconsin-Madison
zyang@chem.wisc.edu
Yen, Chi-Fu
Iowa State University
chifuyen@iastate.edu
Yoo, Jejoong
University of Illinois in Urbana-Champaign
jejoong@gmail.com
Yoshua, Samuel
University of Toronto
sam.yoshua@mail.utoronto.ca
Young, Isaac
Iowa State University
iayoung@iastate.edu
Zeng, Yi
University of Chicago
yizeng@uchicago.edu
47
FACULTY PARTICIPANTS
Name
Institution
Email address
Aksimentiev, Aleksei
University of Illinois in Urbana-Champaign
aksiment@illinois.edu
Chemla, Yann
University of Illinois in Urbana-Champaign
ychemla@illinois.edu
Cheng, Wei
University of Michigan
chengwe@umich.edu
Comstock, Matthew
Michigan State University
mjcomsto@msu.edu
Cornish, Peter
University of Missouri
cornishp@missouri.edu
Galburt, Eric
Washington University in St. Louis
egalburt@biochem.wustl.edu
Goldsmith, Randall
University of Wisconsin-Madison
rhg@chem.wisc.edu
Ha, Taekjip
University of Illinois in Urbana-Champaign
tjha@illinois.edu
Hoskins, Aaron
University of Wisconsin-Madison
ahoskins@wisc.edu
King, Gavin
University of Missouri
kinggm@missouri.edu
Kowalczykowski, Stephen
University of California, Davis
sckowalczykowski@udavis.edu
Lohman, Timothy
Washington University in St. Louis
lohman@biochem.wustl.edu
Meiners, Jens-Christian
University of Michigan
meiners@umich.edu
Ritchie, Kenneth
Purdue University
kpritchie@purdue.edu
Schroeder, Charles
University of Illinois in Urbana-Champaign
cms@illinois.edu
Sivasankar, Sanjeevi
Iowa State University
sivasank@iastate.edu
Spies, Maria
University of Iowa
Maria-spies@uiowa.edu
Wang, Yan Mei
Washington University in St. Louis
ymwang@wustl.edu
Washington, Todd
University of Iowa
todd-washington@uiowa.edu
Yang, Qiong
University of Michigan
qiongy@umich.edu
Yodh, Jaya
University of Illinois in Urbana-Champaign
jyodh@illinois.edu
48
MAPS & INFORMATION
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