Folding

1
Folding

• Judith Klein-SeetharamanDepartment of Structural
  Biology
• jks33_at_pitt.edu

2
Objectives of this Lecture

• Overview Folding/Misfolding
• Anfinsen
• Levinthal Paradox
• Folding Models
• The denatured state
• The molten globule
• Two-state folding
• Deciphering complex folding pathways

3
Objectives of this Lecture

• Overview Folding/Misfolding
• Anfinsen
• Levinthal Paradox
• Folding Models
• The denatured state
• The molten globule
• Two-state folding
• Deciphering complex folding pathways

4
Overview
http//www-nmr.cabm.rutgers.edu/academics/biochem6
94/2006BioChem412/Biochem.412_2-24-2006lecture.pdf

5
Objectives of this Lecture

• Overview Folding/Misfolding
• Anfinsen
• Levinthal Paradox
• Folding Models
• The denatured state
• The molten globule
• Two-state folding
• Deciphering complex folding pathways

6
Anfinsens Experiment
Oznurs slide
Addition of mercaptoethanol and urea
Removal of mercaptoethanol and urea
Native, catalytically active state. Refolded
correctly!
Native, catalytically active ribonuclease A
Unfolded catalytically inactive. Reduced
disulfide bonds.
1/105 random chance
Folding is encoded in the amino acid sequence.
Native state is the minimum energy state.
Anfinsen, 1973.
7
Objectives of this Lecture

• Overview Folding/Misfolding
• Anfinsen
• Levinthal Paradox
• Folding Models
• The denatured state
• The molten globule
• Two-state folding
• Deciphering complex folding pathways

8
How does a protein fold?Levinthals Paradox
Oznurs slide

• Assume a chain of 100 amino acids.
• Allow only 3 conformations.
• - Possible conformations 3100 1048
• Assume bond rotation rate 1014 sec.
• - Reaching the native state would take
• 1026 years ! Longer than the age of
• the universe!

Simplest case random-walk
Energy
Entropy
Protein folding cannot be random-walk.
Dill Chan, 1997
Levinthal, 1968
9
Objectives of this Lecture

• Overview Folding/Misfolding
• Anfinsen
• Levinthal Paradox
• Folding Models
• The denatured state
• The molten globule
• Two-state folding
• Deciphering complex folding pathways

10
The Three Protein Folding Models
Oznurs slide
Framework model
Hydrophobic collapse model
Nucleation condensation model
http//www.makro.ch.tum.de/users/BFHZ/Scheibel/Sch
eibel20200320Bordeaux-1.pdf
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Objectives of this Lecture

• Overview Folding/Misfolding
• Anfinsen
• Levinthal Paradox
• Folding Models
• The denatured state
• The molten globule
• Two-state folding
• Deciphering complex folding pathways

12
Random Coil and Denatured State
Oznurs slide
Florys isolated pair hypothesis
Rg values determined by SAXS
F,? angles of each residue is sterically
independent There should not exist any non-local
interactions.
Rg values of 28 denatured proteins obeys the
Florys power law.
Rg RgNv N Length (Residues)
v Solvent viscosity parameter
Sosnick, T.R., et al. 2004
Flory, 1969.
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Testing the random coil statistics
Oznurs slide
For a protein 8 of the residues are varied the
remaining 92 of the residues remained fixed in
their native conformation.
33 proteins
Number of residues
Simulated Rg follows the power law.
Despite 92 of the native structure kept, random
coil statistics are obtained.
Fitzkee, N.C. and Rose, G.D. 2004
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The Denatured StateDoes Florys hypothesis hold?
Oznurs slide
Conformations of polyalanine chains are
enumerated to test the hypothesis.
A,G,M,R,L,F,E,K,Q J,P,O,I,o
Florys hypothesis is not valid for polypeptide
chains. Backbone conformations are limited by
additional steric clashes.
Pappu et.al 2003.
15
Which NMR spectrum is of folded and which is of
unfolded lysozyme?
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Which NMR spectrum is of folded and which is of
unfolded lysozyme?
folded
unfolded
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How would you use NMR to test for residual
structure?
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How would you identify residual structure in
unfolded proteins with NMR?

• What types of NMR parameters do you know?
• chemical shifts
• coupling constants
• HetNOE
• longitudinal relaxation rates (R1)
• transverse relaxation rates (R2)

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How would you identify residual structure in
unfolded proteins with NMR?

• 1. Measurement of NMR parameters in 15N-labeled
  unfolded protein
• chemical shifts
• coupling constants
• HetNOE
• longitudinal relaxation rates (R1)
• transverse relaxation rates (R2)
• 2. Comparison of NMR parameters with random coil
• 3. Deviation from random coil identifies residual
  structure
• Application to unfolded conformations of hen egg
  white lysozyme
• oxidized in 8M urea
• reduced and methylated in 8M urea
• reduced and methylated in water

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Chemical shift differences between unfolded
lysozyme and random coil
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Dynamics in folded/unfolded lysozyme
Unfolded
Arrows indicate oxidized (all disulfide bonds
present) lysozyme
Folded
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Relaxation Rates in Unfolded Lysozyme
Unfolded lysozyme can be studied in 8 M
urea. Unfolded lysozyme can also be studied
without urea, if the disulfide bonds are reduced
and the cysteines are derivatized to prevent them
from forming disulfide bonds.
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Relaxation Rates in Unfolded Lysozyme
What do you observe?
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Relaxation Rates in Unfolded Lysozyme
Regions with higher relaxation rates are
localized as clusters. ? Presence of clusters of
residual structure that are restricted in
conformational space, thus relax faster.
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How would you analyze the relaxation data?
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What are the assumptions of the model-free
approach?
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Analysis of the relaxation data

• Three means of analysis have been proposed
• Model-free approach
• Cole-Cole distributions
• Gaussian clusters

However What gives rise to these clusters is not
known.
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Relaxation Rates in Unfolded Lysozyme
There are six clusters of residual structure in
HEWL-SME.
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Mapping of residual structure on the native
structure
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Hydrophobic clusters of residual structure
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What stabilizes the clusters of residual
structure?
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What stabilizes the clusters of residual
structure?

• Long-range interactions?
• Local structure?
• How would you test this?

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Approach 1

• Peptides if peptides without structural context
  of the full chain contain structure, then this
  structure is independent of long-range
  stabilization

34
Approach 2

• Test for the presence of long-range interactions
  in the context of the full-length protein
• What approaches can you imagine to test for
  long-range interactions?

35
Residual Structure Mapped onto Native Structure
Clusters of deviations from random coil dynamics
map onto proximal regions in the native
structure, except cluster 3.
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How would you test for the presence of long-range
interactions?Approach 1. Study effect of mutation
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Effect of mutation on chemical shifts
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Effect of mutation on relaxation rates
A single point mutation, W62G in cluster 3,
disrupts all clusters in reduced and methylated
lysozyme.
39
Effect of mutation on chemical shifts
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Effect of mutation on relaxation rates
41
Model for unfolded ensemble
42
Compactness by NMR
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Approach 2. FRET
- So far has been only used for global changes,
not to detect specific contact formation
Haustein and Schwille (2004) Current Opin.
Structural Biology 14, 531-540.
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Approach 3. EPR proton relaxation
interaction up to 20-25Å
Staphylococcus nuclease Gillespie and Shortle
(1997) JMB 268, 170-184 and 158-169.
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Role of disulfide bonds for dynamics
Disulfide bonds and hydrophobic clusters are
cooperative.