Shaobin Guo
11/20/2012
various types of Single-molecule force
spectroscopy
• Optical tweezers
• Magnetic tweezers
• Atomic force microscopy (AFM)
• Micro-needle manipulation
• Biomembrane force probe
• Flow-induced stretching
Single-molecule manipulation
capacity
• Length: 10
- 10-4 m / measurement of
RNA polymerase advancing a single
base pair to manipulation of cells
• Force: 10
-10
-14
- 10-8 Newton / assaying
nucleic acid folding kinetics to
mechanical disruption of covalent
bonds
Optical tweezers (Trap)
• Lasers: near-infrared wavelengths
(800-1100
nm)
• High numerical aperture (NA) microscope
objective (at least 1.2)
• An approximate linear spring for small
displacements (~150 nm)
• Position detection: back-focal plane (BFP)
interferometry
• Calibration: position detector and trap
applications
Infrared laser
silica bead
kinesin molecule
microtubule
Interaction
assay
Infrared laser
RNA polymerase molecule
Tethered
assay
Infrared laser
Dumbbell
assay
Limitations and Drawbacks
• Require optically homogeneous preparations
and highly purified samples for high
resolution trapping.
• Lack selectivity and exclusivity
• Local heating
• Optical damage
• Limited range of applied force (0.1-100 pN)
and range of displacement (~150 nm or less)
magnetic tweezers
• A pair of permanent magnets
• Inverted microscope with a charge-coupled device (CCD)
• Able to rotate super-paramagnetic beads ranging from 0.5
to 5 um
• Force-clamp property: an effective stiffness on the order of
10-6 pN/nm
• Free from sample heating, photodamage and other
problems related to optical tweezers
• Drawbacks: not versatile, unable to directly measure
rotational torque generation, limited to slow and large
displacements
Electromagnetic
tweezers
• Sharp electromagnetic tips
• Able to exert forces in excess of 1 nN
• Easy to control force and rotation by
changing current
• Three-dimensional position control
• Drawbacks: cumbersome feedback control,
sophisticated pole pieces, lack of sensitivity
and heating.
Atomic Force microscopy (AFM)
• A very high-resolution type of scanning
probe microscopy, with demonstrated subnm resolution
• Able to measure inter- and intramolecular
interaction forces with pN resolution
• Simple and rapid sample preparation, and
the ability to conduct measurements under
near-physiological conditions
applications
• Study of molecular bond’s rupture: covalent
bonds to protein unfolding
• Force-clamp spectroscopy by AFM
• Investigation of supramolecular assemblies
• Combination of AFM imaging with force mapping
and spectroscopy to image the surface topology
at high resolution and measure the unbinding or
unfolding forces at well-defined locations.
• High-speed AFM which makes it possible to
follow molecular events in real time.
Limitations and Drawbacks
• Narrow useful force range resulting
from large size and relative high
stiffness of the cantilevers
• Lack of specificity
Background
• Regiospecific thiol-disulfide exchange
reactions: electrons are reshuffled between a
thiolate and a disulfide bond via an SN2
mechanism that produces a different
disulfide and a new thiolate
problems with bulk techniques
• Inability to differentiate disulfide isomers
• Variations among different techniques
used to analyze reactions and
subsequent divergent interpretations
• Interference from the reverse reaction
experiment set-up
Cantileve
r
Tip
Force: 250
pN
Piezoelectric
scanner
Reduction events from
polypeptides containing single
disulfide bond
14.5
nm
(I2732–
75)
8
Predicted
extension after
the reduction of a
disulfide bond
10
nm
(I2724–
55)
8
Reduction events from
polypeptides containing two
disulfide bonds
(I272S-S)4
Cleavage of disulfide 32-75 in
2S-S
I27
Pathways available for the reduction of
disulfide 24–55 after the reduction of
disulfide 32–75
At high concentrations of L-Cys the frequency of
the 10 nm steps increases at the expense of the 3
and 8nm steps
Thanks!
Questions?