Folding

1. Folding Judith Klein-SeetharamanDepartment of Structural Biology jks33@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 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 Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

4. Overview http://www-nmr.cabm.rutgers.edu/academics/biochem694/2006BioChem412/Biochem.412_2-24-2006lecture.pdf 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 Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

6. 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. 1/105 random chance Folding is encoded in the amino acid sequence. Native state is the minimum energy state. Anfinsen, 1973. 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 Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

8. Oznur’s slide: How does a protein fold?Levinthal’s Paradox • 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 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 Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

10. Oznur’s slide: The Three Protein Folding Models Framework model Hydrophobic collapse model Nucleation condensation model http://www.makro.ch.tum.de/users/BFHZ/Scheibel/Scheibel%202003%20Bordeaux-1.pdf 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 Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

12. 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. Rg values of 28 denatured proteins obeys the Flory’s power law. • Rg= RgNv • N = Length (Residues) • v = Solvent viscosity parameter Sosnick, T.R., et al. 2004 Flory, 1969. Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

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

14. Oznur’s slide: The Denatured StateDoes 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} Flory’s hypothesis is not valid for polypeptide chains. Backbone conformations are limited by additional steric clashes. Pappu et.al 2003. Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

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

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

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

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

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

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

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

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

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

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

28. 3. 2. 5. 4. 6. 1. Random Coil Model of Segmental Motion + Gaussian Distributions of Deviations 2 - - | i x | | i j | N 0 - - å å = + l R ( i ) R e Ae b int rinsic = j 1 x 0 Relaxation Rates in Unfolded Lysozyme There are six clusters of residual structure in HEWL-SME. Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

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

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

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

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

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

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

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

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

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

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

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

43. 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. 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, 170-184 and 158-169. Molecular Biophysics III – Klein-Seetharaman – Folding Lecture

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