1. Judith Klein-Seetharaman Department of Structural Biology
Folding
Judith Klein-Seetharaman Department of Structural Biology

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
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

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
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

4. Molecular Biophysics III – Klein-Seetharaman – Folding Lecture
Overview
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

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
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

6. Anfinsen’s Experiment
Oznur’s slide:
Anfinsen’s Experiment
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.
[2] Anfinsen, C. (1973). Principles that govern the folding of protein chains. Science
Ribonuclease A (RNaseA) is an extracellular enzyme of 124 residues with four disulfide bonds. In the first phase of the experiment, the S-S bonds were reduced to eight -SH groups (using mercaptoethanol, HS-CH2-CH2-OH); the protein was then denatured with 8 M urea. Under these conditions, the enzyme is inactive and becomes a flexible random polymer. In the second phase, the urea was slowly removed (dialysis); then the the -SH groups were oxidized back to S-S bonds. If the protein was able to regain its native structure spontaneously after removal of the urea, we expect that it would also regain its activity. In fact, the activity was >90% of the untreated enzyme. Moreover, sequence analysis showed that nearly all of the correct S-S bonds had been formed.
There are exceptions to this, but these appear to be the cases where metastable states are trapped kinetically during folding.(alpha-lyctic protease , Sohl 1998 Nature)
1/105 random chance
Folding is encoded in the amino acid sequence. Native state is the minimum energy state.
Anfinsen, 1973.
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

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
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

8. How does a protein fold? Levinthal’s Paradox
Oznur’s slide:
How does a protein fold? Levinthal’s Paradox
Assume a chain of 100 amino acids.
Allow only 3 conformations.
- Possible conformations = ~ 1048
Assume bond rotation rate sec.
- Reaching the native state would take:
1026 years ! Longer than the age of
the universe!
Simplest case: random-walk
Energy
Ribonuclease A (RNaseA) is an extracellular enzyme of 124 residues with four disulfide bonds. In the first phase of the experiment, the S-S bonds were reduced to eight -SH groups (using mercaptoethanol, HS-CH2-CH2-OH); the protein was then denatured with 8 M urea. Under these conditions, the enzyme is inactive and becomes a flexible random polymer. In the second phase, the urea was slowly removed (dialysis); then the the -SH groups were oxidized back to S-S bonds. If the protein was able to regain its native structure spontaneously after removal of the urea, we expect that it would also regain its activity. In fact, the activity was >90% of the untreated enzyme. Moreover, sequence analysis showed that nearly all of the correct S-S bonds had been formed.
There are exceptions to this, but these appear to be the cases where metastable states are trapped kinetically during folding.(alpha-lyctic protease , Sohl 1998 Nature)
Entropy
Protein folding cannot be random-walk.
Dill & Chan, 1997
Levinthal, 1968
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

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
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

10. The Three Protein Folding Models
Oznur’s slide:
The Three Protein Folding Models
Framework
model
Hydrophobic collapse
model
Typically, the unfolded state is depicted as a rugged energy landscape with an exorbitant number of local
minima. Under suitable conditions, the protein negotiates thi landscape spontaneously and finds its way to the global minimum—
the native state.
Understanding the mechanism of protein folding requires detailed structural characterization
of all of the species formed during the folding reaction, including the native, partially folded, and fully denatured states.
Nucleation condensation
model
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

11. 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
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

12. Random Coil and Denatured State
Oznur’s slide:
Random Coil and Denatured State
Flory’s isolated pair hypothesis
Rg values determined by SAXS
“Φ,Ψ angles of each residue is sterically independent”
There should not exist any non-local interactions.
Flory, P. J. (1969) Statistical Mechanics of Chain Molecules (Wiley, New York).
Sosnick, T.R., et al Random-coil behavior and the dimensions of chemically unfolded proteins. Proc. Natl. Acad. Sci. 101: 12491–12496
the mainchain trace of the
polypeptide can be described in terms of a series of
virtual bonds (or peptide planes) connecting the C
atoms (Fig. 1) [21]. The relative orientation of two of
these virtual bonds (i and i+1) is determined by the pair
of torsion angles i and i defining rotation about the
bonds adjoining the C atom of residue i.
Ramachandran map was computed by generating 150,000 independent
conformations within each mesostate by using backbone-dependent
values for the N–Ca–C9 bond angle (Table 1).
tigger.uic.edu/.../ANJUM05/psi_phi_angles.jpg
Rg values of 28 denatured proteins
obeys the Flory’s power law.
Rg= RgNv
N = Length (Residues)
v = Solvent viscosity parameter
Flory, 1969.
Sosnick, T.R., et al. 2004
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

13. Testing the random coil statistics
Oznur’s slide:
Testing the random coil statistics
For a protein ≈8% of the residues are varied; the remaining ≈92% of the residues remained fixed in their native conformation.
33 proteins
Fitzkee, N.C. and Rose, G.D Reassessing random-coil statistics in unfolded proteins. Proc. Natl. Acad. Sci. 101: 12497–12502
Flory showed (ref. 4, page 43) that the radius of gyration, RG, follows a simple scaling law:
[1]where N is the number of residues, R0 is a constant related to persistence length, and ν is the scaling factor of interest that depends on solvent quality.
Yet, this physically absurd model (in which 92% of the native structure is retained) successfully reproduces random-coil statistics for RG and L2 in good solvent (e.g., 6 M guanidinium chloride). Therefore, it is not too surprising that transient organization in denatured proteins could also give rise to the random-coil statistics observed in experiment (10).
Compactness is one of the important properties
pertinent to the structure of protein molecules in
solution, and it also characterizes the degree of
folding. Small-angle X-ray scattering (SAXS) is one
of the few techniques that provides a direct
measurement of compactness, since SAXS gives
overall geometric information such as the size and
shape of the molecule.
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
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

14. The Denatured State Does Flory’s hypothesis hold?
Oznur’s slide:
The Denatured State Does Flory’s hypothesis hold?
Conformations of polyalanine chains are enumerated to test the hypothesis.
+ ={A,G,M,R,L,F,E,K,Q}
* = {J,P,O,I,o}
Ramachandran map was computed by generating 150,000 independent
conformations within each mesostate by using backbone-dependent
values for the N–Ca–C9 bond angle (Table 1).
. Backbone dihedral angles in
proteins of known structure lie well inside the allowed regions of
a f,cmap, .
If each f,c pair is independent (3), constraints that sterics
impose on the dipeptide are insufficient to limit the conformations
accessible to a peptide backbone, even a short one.
In contrast, our analysis of short polyalanyl chains shows that
Flory’s hypothesis is not valid for polypeptide chains. Backbone conformations are limited by additional steric clashes.
Pappu et.al 2003.
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

15. Which NMR spectrum is of folded and which is of unfolded lysozyme?
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

16. Which NMR spectrum is of folded and which is of unfolded lysozyme?
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

17. How would you use NMR to test for residual structure?
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

18. Molecular Biophysics III – Klein-Seetharaman – Folding Lecture
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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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

19. Molecular Biophysics III – Klein-Seetharaman – Folding Lecture
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
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

20. Chemical shift differences between unfolded lysozyme and random coil
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

21. Dynamics in folded/unfolded lysozyme
Arrows indicate oxidized (all disulfide bonds present) lysozyme
Folded:
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

22. 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.
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

23. Relaxation Rates in Unfolded Lysozyme
What do you observe?
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

24. 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.
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

25. How would you analyze the relaxation data?
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

26. What are the assumptions of the model-free approach?
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

27. 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.
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

28. Relaxation Rates in Unfolded Lysozymeå = - 1 |
int ) ( N j i
rinsic e R l
Random Coil Model of Segmental Motion å - + 2 | x b i Ae
+ Gaussian Distributions of Deviations 1. 2. 3. 4. 5. 6.
There are six clusters of residual structure in HEWL-SME.
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

29. Mapping of residual structure on the native structure
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

30. Hydrophobic clusters of residual structure
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

31. What stabilizes the clusters of residual structure?
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

32. What stabilizes the clusters of residual structure?
Long-range interactions?
Local structure?
How would you test this?
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

33. Molecular Biophysics III – Klein-Seetharaman – Folding Lecture
Approach 1
Peptides: if peptides without structural context of the full chain contain structure, then this structure is independent of long-range stabilization
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

34. Molecular Biophysics III – Klein-Seetharaman – Folding Lecture
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?
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

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.
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

36. Molecular Biophysics III – Klein-Seetharaman – Folding Lecture
How would you test for the presence of long-range interactions? Approach 1. Study effect of mutation
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

37. Effect of mutation on chemical shifts
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

38. Effect of mutation on relaxation rates
A single point mutation, W62G in cluster 3, disrupts all clusters in reduced and methylated lysozyme.
11/24/2018
Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

39. Effect of mutation on chemical shifts
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

40. Effect of mutation on relaxation rates
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

41. Model for unfolded ensemble
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

42. Molecular Biophysics III – Klein-Seetharaman – Folding Lecture
Compactness by NMR
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

43. Molecular Biophysics III – Klein-Seetharaman – Folding Lecture
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,
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

44. Approach 3. EPR – proton relaxation
interaction up to 20-25Å
Staphylococcus nuclease – Gillespie and Shortle (1997) JMB 268, and
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

45. Role of disulfide bonds for dynamics
A. Wild-Type
B. W62G
Disulfide bonds and hydrophobic clusters are cooperative.
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Molecular Biophysics III – Klein-Seetharaman – Folding Lecture