MCB Exam III Review
Ji Woong Park
December 13th, 2014
Material Coverage
• My review covers 45 points. It’s lectures by Stewart,
Huettner, Weihl, Amarasinghe, and Fremont.
• Due to the diverse range of topics (cancer to
crystallography), you may need to use slides not in the
review to help your understanding.
• But, the exam questions will be based on the review
slides. There will be, however, some questions that
require solid understanding/application to answer
them (hence, the above point)
• Previous years’ exams will be helpful but it’s incorrect
to assume they will be like Exam II.
Terms To Know for “Application” Questions
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RT-qPCR/RNAi/Knockout Gene or Mice
Western Blot/IP/Co-IP
Transfection/Infection
Electrophysiology
Mass Spec/NMR/Crystallography
Site-Directed Mutagenesis
Inhibitors/Dominant Negatives
Fluorescence/Viability/Toxicity Assays
Telomere Function
distinguishes between the chromosome end and a double strand break
protects the chromosome from end-to-end fusions
An emerging paradigm: the telomere complex
does not exclude DNA surveillance, repair and
replication machinery; rather it directs, modulates
and specializes the activities of these proteins to
ensure high fidelity replication and telomere
stability
Old view
TIN2
TPP1
POT1POT1
RAP1
DNA repair
machinery
New view
DNA repair/replication
machinery
The “telomere” hypothesis
Telomere Length
Population Doubling
Senescence
1
2
3
4
Time
5
6
The telomere hypothesis
p53
Rb
Telomere Length
Stop
Time
Senescence
The telomere hypothesis
Telomere Length
p53
Rb
Continued
proliferation
Crisis
telomere dysfunction
genetic catastrophe
cell death
Time
Telomerase adds telomeric repeats
to the 3’ termini of the chromosome
-only the catalytic (hTERT) and RNA (hTR) components
are required for activity in vitro
hTERT
hTR
CAAUCCCAAUC
The telomere hypothesis
Telomere Length
ALT
Stop
Senescence
p53
Rb
Crisis
hTERT
1 in ~107
Stable telomere maintenance
Time
Telomere Length
The telomere hypothesis
ALT
Stop
Crisis
hTERT
1 in ~107
Time
ALT
Telomerase +
Telomerase allows ALT cells to form tumors
hTERT
H-ras
TUMORS
GM847
Immortal
Expresses SV40 Early region
Lacks telomerase
Stewart et al 2002
Extra-telomeric functions of hTERT
Overexpression of mTERT in murine models increases tumor rates
in aged mice
Overexpression of hTERT results in resistance to apoptosis
Ectopic expression of mTERT in skin results “hairy” mice, increased
stem cell pool
Telomerase is favored over ALT in human tumors
hTERT expression is required in normal fibroblasts to avoid
senescence
Time/Stress
Senescence
Senescence evasion
Stromal Promotion
Normal
ECM
Epithelial cell
Premalignant
Altered ECM
Endothelial cell
Preneoplastic cell
Tumor
Young fibroblast
Senescent fibroblast
Cancer cell
Immune cell
Senescent epithelial cell
For additional reading (highly recommend)
Types of Stem Cells
Embryonic – from the inner cell mass of preimplantation embryos, prior to formation of
the 3 germ layers (ectoderm, mesoderm,
endoderm)
Somatic – undifferentiated cells found in specific
locations in “mature” tissues
iPS cells – induced pluripotent stem cells
generated by reprogramming differentiated
cells (or cell nuclei, i.e. therapeutic cloning)
Reprogramming
• SCNT – somatic cell nuclear transfer (reproductive and
therapeutic cloning) – deterministic and fairly rapid
• iPS – induced pluripotent stem cells – slow and
stochastic (until recently)
• Transdifferentiation – conversion of one terminally
differentiated cell type into another without dedifferentiation to an immature phenotype. Must rule
out cell fusion or other explanations.
Generating iPS cells
• Express transcription factors:
Oct3/4, Sox2, Klf4 and c-Myc (OSKM)
Oct3/4, Sox2, Nanog and Lin28
• Initial de-differentiation and proliferation
(day 1-3, enhanced by Myc); histone modification and
chromatin reorganization
• 2nd wave of gene expression - stem cell and
development related genes (day 9-12); DNA
demethylation and X reactivation
OR
Transdifferentiation
• Conversion from one differentiated cell type
to another without evident de-differentiation
and re-differentiation
• Must not be confused by cell fusion or
selection for rare pluripotent cells in the
source material.
• Induced by expression of transcription factors
and microRNAs
Protein Degradation in the Cell
Ub
Autophagy
Nucleus
Aggresome
Ub
UPS
Ub
Ub
Endocytosis
Protein Degradation
 Ubiquitin/Proteasome Pathway
80-90%
Most intracellular proteins
• Lysosomal processes
10-20%
Extracellular proteins
Cell organelles
Some intracellular proteins
UBIQUITIN
 Small peptide that is a “TAG”
 76 amino acids
 C-terminal glycine - isopeptide
bond with the e-amino group of
lysine residues on the substrate
 Attached as monoubiquitin or
polyubiquitin chains
G
K
Ubiquitination of proteins is a FOUR-step process
 First, Ubiquitin is activated by forming
a link to “enzyme 1” (E1).
 Then, ubiquitin is transferred to one
of several types of “enzyme 2” (E2).
 Then, “enzyme 3” (E3) catalizes the
transfer of ubiquitin from E2 to a Lys
e-amino group of the “condemned”
protein.
 Lastly, molecules of Ubiquitin are
commonly conjugated to the protein to
be degraded by E3s & E4s
AMP
The proteasomal DUB Usp14 impairs
protein degradation
Lee, BH et al
Nature 467:179-84
2010
Autophagy
• Lysosomal degradation of proteins and
organelles
• Occurs via three routes
– Macroautophagy
– Microautophagy (direct uptake of cellular debris
via the lysosome)
– Chaperone mediated autophagy (selective import
of substrates via Hsc70 and Lamp2a)
Selective Autophagy
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Aggregaphagy– p62/SQSTM1, Nbr1
Mitophagy – Parkin, Nix
Reticulophagy – endoplasmic reticulum
Ribophagy – translating ribosomes
Xenophagy – e.g. Salmonella via optineurin
Lipophagy – autophagy mediated lipolysis
• Performed by an expanding group of ubiquitin
adaptors
Rapamycin as an inducer of
autophagy
 Immunosuppressant used to treat transplant rejection
 Inhibits the mTOR pathway
 mTOR integrates extrinsic growth signals and cellular
nutrient status and energy state
 Active mTOR
 Protein synthesis and cell growth
 Inactive mTOR (or rapamycin treatment)
 Inhibition of protein synthesis and increased autophagic
degradation of protein
Protein Structures from an NMR Perspective
Analyzing NMR Data is a Non-Trivial Task!
there is an abundance of data that needs to be interpreted
X
Not A Direct Path!
Interpreting NMR Data Requires
Making Informed “Guesses” to
Move Toward the “Correct” Fold
Distance from Correct Structure
Initial rapid convergence to
approximate correct fold
Correct structure
NMR Data Analysis
Iterative “guesses” allow
“correct” fold to emerge
Protein Structure Determination
by NMR
•Stage I—Sequence specific resonance
assignment
•State II – Conformational restraints
•Stage III – Calculate and refine structure
Why use deuteration?
• What are the advantages?
• What are the disadvantages?
NMR Structure Determination
NOE
NOE
- a through space correlation (<5Å)
- distance constraint
4.1Å
2.9Å
Coupling Constant (J)
- through bond correlation
J
NH
CaH
- dihedral angle constraint
Chemical Shift
- very sensitive to local changes
in environment
- dihedral angle constraint
Dipolar coupling constants (D)
- bond vector orientation relative
to magnetic field
- alignment with bicelles or viruses
CaH
D
NH
Analysis of the Quality of NMR Protein Structures
Is the “Average” NMR Structure a Real Structure?
• No-it is a distorted structure
level of distortions depends on the similarity between the structures in the
ensemble
 provides a means to measure the variability in atom positions between an
ensemble of structures
Expanded View of an “Average” Structure
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Some very long,
stretched bonds
Position of atoms are so
scrambled the graphics
program does not know which
atoms to draw bonds between
Some regions of the structure
can appear relatively normal
An 7-step program for protein structure
determination by x-ray crystallography
1. Produce monodisperse protein either alone or as relevant complexes
2. Grow and characterize crystals
3. Collect X-ray diffraction data
4. Solve the phase problem either experimentally or computationally
5. Build and refine an atomic model using the electron density map
6. Validation: How do you know if a crystal structure is right?
7. Develop structure-based hypothesis
1.
Produce monodisperse protein
either alone or as relevant complexes
Methods to determine protein purity, heterogeneity, and monodispersity
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Gel electrophoresis (native, isoelectric focusing, and SDS-PAGE)
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Size exclusion chromatography
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Dynamic light scattering http://www.protein-solutions.com/
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Circular Dichroism Spectroscopy http://www-structure.llnl.gov/cd/cdtutorial.htm
Characterize your protein using a number of biophysical methods
Establish the binding stoichiometry of interacting partners
2. Grow and characterize crystals
Hanging Drop vapor diffusion
Sitting drop, dialysis, or under oil
Macro-seeding or micro-seeding
Sparse matrix screening methods
Random thinking processes, talisman, and luck
The optimum conditions for crystal nucleation are not necessarily
the optimum for diffraction-quality crystal growth
Hanging Drop
Sitting drop
Commercial screening kits available from
http://www.hamptonresearch.com;
http://www.emeraldbiostructures.com
Space Group P21
4 M3 /ASU
diffraction >2.3Å
14.4% Peg6K
NaCacodylate pH 7.0
200mM CaCl2
Space Group C2
2 M3 /ASU
diffraction >2.1Å
18% Peg4K
Malic Acid/Imidazole pH 5.1
100mM CaCl2
Space Group P3121
3 M3 + 3 MCP-1/ASU
diffraction > 2.3Å
18% Peg4K
NaAcetate pH 4.1
100mM MgCl2
No Xtals?
Decrease protein heterogeneity
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Remove purification tags and other artifacts of protein production
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Remove carbohydrate residues or consensus sites (i.e., N-x-S/T)
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Determine domain boundaries by limited proteolysis followed by mass
spectrometry or amino-terminal sequencing. Make new expression constructs if
necessary.
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Think about the biochemistry of the system! Does your protein have cofactors, accessory proteins, or interacting partners to prepare as complexes? Is
their an inhibitor available? Are kinases or phosphatases available that will allow
for the preparation of a homogeneous sample?
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Get a better talisman
3. Collect X-ray diffraction data
Initiate experiments using home-source x-ray generator and detector
Determine liquid nitrogen cryo-protection conditions to reduce crystal decay
While home x-rays are sufficient for some questions, synchrotron radiation is preferred
Anywhere from one to hundreds of crystals and diffraction experiments may be required
Argonne National Laboratory Structural Biology Center beamlineID19
at the Advanced Photon Source http://www.sbc.anl.gov
4. Solve the phase problem either
experimentally or computationally
Structure factor equation:
By Fourier transform we can obtain the electron density.
We know the structure factor amplitudes after successful data collection.
Unfortunately, conventional x-ray diffraction doesn’t allow for direct phase measurement.
This is know as the crystallographic phase problem.
Luckily, there are a few tricks that can be used to obtain estimates of the phase a(h,k,l)
Experimental Phasing Methods
MIR - multiple isomorphous replacement - need heavy atom incorporation
 MAD - multiple anomalous dispersion- typically done with SeMet replacement
MIRAS - multiple isomorphous replacement with anomalous signal
SIRAS - single isomorphous replacement with anomalous signal
Computational Methods
MR - molecular replacement - need related structure
Direct and Ab Initio methods - not yet useful for most protein crystals
MAD phasing statistics for the AP-2 a-appendage
5. Build an atomic model using the electron density map
Electron density for the AP-2 aappendage
Initial bones trace for the AP-2 aappendage
Final trace for the AP-2 aappendage
The resolution of the electron-density map
and the amount of detail that can be seen
Resolution
Structural Features Observed
5.0 Å
3.5 Å
3.0 Å
2.8 Å
2.5 Å
Overall shape of the molecule
Ca trace
Side chains
Carbonyl oxygens (bulges)
Side chain well resolved,
Peptide bond plane resolved
Holes in Phe, Tyr rings
Current limit for best protein
crystals
1.5 Å
0.8 Å
6. Validation: How do you know if a crystal structure is right?
The R-factor
R = S(|Fo-Fc|)/S(Fo)
where Fo is the observed structure factor amplitude and Fc is calculated using the atomic model.
R-free
An unbiased, cross-validation of the R-factor. The R-free value is calculated with typically 5-10% of the observed reflections which are set aside from atomic refinement calculations.
Main-chain torsions: the Ramachandran plot
Geometric Distortions in bond lengths and angles
Favorable van der Waals packing interactions
Chemical environment of individual amino acids
Location of insertion and deletion positions in related sequences
7. Develop structure-based hypothesis
Structure-Based Mutagenesis of the a-appendage
Traub LM, Downs MA, Westrich JL, and Fremont DH: (1999) Crystal structure of the
a-appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly. Proc.
Natl. Acad. Sci. U.S.A. 96:8907-8912.
Selection of E16 specific epitope variants of DIII
Yeast library of DIII variants
created by error prone PCR
E -DIII
Pooled
DIII
mAbs
E16 staining
DIII mutations at Ser306, Lys307, ThrE330 and Thr332 significantly diminish E16 binding
GOOD LUCK – Interview season is coming!