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Protein Folding Energetics, Kinetics and Models

Protein Folding Energetics, Kinetics and Models. Oznur Tastan oznur@cs.cmu.edu Graduate Student Carnegie Mellon University. Lecture Outline. Introduction: What is protein folding and why it is a problem? Globular Protein Folding Models

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Protein Folding Energetics, Kinetics and Models

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  1. Protein FoldingEnergetics, Kinetics and Models Oznur Tastan oznur@cs.cmu.edu Graduate Student Carnegie Mellon University

  2. Lecture Outline • Introduction: What is protein folding and why it is a problem? • Globular Protein Folding Models • Detection and characterization of denatured states, intermediates, such as the molten globule and comparison to folded states • Kinetics and pathways • Membrane Protein Folding Models • 2-stage and 3-stage hypothesis • New long range interaction hypothesis • Summary Molecular Biophysics III: Spring 2006: Oznur Tastan

  3. Lecture Outline • Introduction: What is protein folding and why it is a problem? • Globular Protein Folding Models • Detection and characterization of denatured states, intermediates, such as the molten globule and comparison to folded states • Kinetics and pathways • Membrane Protein Folding Models • 2-stage and 3-stage hypothesis • New long range interaction hypothesis • Summary Molecular Biophysics III: Spring 2006: Oznur Tastan

  4. Our focus in this lecture http://www-nmr.cabm.rutgers.edu/academics/biochem694/2006BioChem412/Biochem.412_2-24-2006lecture.pdf Molecular Biophysics III: Spring 2006: Oznur Tastan

  5. Anfinsen’s Experiment Addition of mercaptoethanol and urea Removal of mercaptoethanol and urea Native, catalytically active state. Refolded correctly! Native, catalytically active state. 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: Spring 2006: Oznur Tastan

  6. The Protein Folding Problem:Writing the book of Protein Origami Now collapse down hydrophobic core, and fold over Helix A to the dotted line. Bring charged residues of ‘A’ into close proximity of ‘B’ ..? +membrane http://www.idi.ntnu.no/grupper/KS-grp/microarray/slides/drablos/Fold_recognition/sld006.htm Molecular Biophysics III: Spring 2006: Oznur Tastan

  7. 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: Spring 2006: Oznur Tastan

  8. Why is protein folding problem difficult? • Folding can be very fast, millisecond to second (slow folding is easier) • Small energy changes between the denatured state to the native state ( 1-15 kcal/mol) - equivalent to the strength of a few hydrogen bonds • The states populated along pathway are ensembles of structures Comparison from multiple complementary techniques are required. Molecular Biophysics III: Spring 2006: Oznur Tastan

  9. 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: Spring 2006: Oznur Tastan

  10. Lecture Outline • Introduction: What is protein folding and why it is a problem? • Globular Protein Folding Models • Detection and characterization of denatured states, intermediates, such as the molten globule and comparison to folded states • Kinetics and pathways • Membrane Protein Folding Models • 2-stage and 3-stage hypothesis • New long range interaction hypothesis • Summary Molecular Biophysics III: Spring 2006: Oznur Tastan

  11. The Native State • A complex balance between: 1) Short-range local interactions -intrinsic conformational preferences of the amino acids 2) Medium-range interactions -stabilizing regions of secondary structure 3) Long-range interactions -tertiary interactions determining the global fold • Generally single conformation (with small fluctuations around the mean torsion angles). Molecular Biophysics III: Spring 2006: Oznur Tastan

  12. 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: Spring 2006: Oznur Tastan

  13. 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: Spring 2006: Oznur Tastan

  14. 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: Spring 2006: Oznur Tastan

  15. Can we get a structure of the “denatured state”? • When the folded state breaks down: • The dispersion of all resonances decreases: • - Extensive overlap of peaks. • 2. Greater dynamic motions between residues: • - Weak or eliminated NOEs between protons. • 3. Ensemble of conformations: • -Each NMR parameter reflects an average over a dynamic ensemble of conformations. Attainment of a high-resolution structure is not possible in the non-native state. Molecular Biophysics III: Spring 2006: Oznur Tastan

  16. NMR as a tool to study denatured states Unfolded lysozyme: Folded lysozyme: For small proteins, all backbone resonances can be resolved even in the denatured state. Molecular Biophysics III: Spring 2006: Oznur Tastan

  17. Which and how do we use NMR parameters? 1. Measurement of NMR parameters in 15N-labeled unfolded protein 2. Comparison of NMR parameters - unfolded with random coil parameters (sources: - statistical analysis from unfolded peptides - random coil models (e.g. polymer model, Model-free analysis, Flory etc.) - unfolded with folded state parameters - different degrees of unfolded states • Chemical shifts • Relaxation rates • Heteronuclear NOE • Dipolar couplings • Scalar couplings Molecular Biophysics III: Spring 2006: Oznur Tastan

  18. Which and how do we use NMR parameters? 1. Measurement of NMR parameters in 15N-labeled unfolded protein 2. Comparison of NMR parameters - unfolded with random coil parameters (sources: - statistical analysis from unfolded peptides - random coil models (e.g. polymer model, Model-free analysis, Flory etc.) - unfolded with folded state parameters - different degrees of unfolded states • Chemical shifts • Relaxation rates • Heteronuclear NOE • Dipolar couplings • Scalar couplings Molecular Biophysics III: Spring 2006: Oznur Tastan

  19. Persistence of native-like topology in the denatured states SNase N-H Dipolar couplings Unfolded Denatured proteins can preserve long range ordering, in conflict with the random-coil models. Folded Shortie. et. al. 2001 Molecular Biophysics III: Spring 2006: Oznur Tastan

  20. Which and how do we use NMR parameters? 1. Measurement of NMR parameters in 15N-labeled unfolded protein 2. Comparison of NMR parameters - unfolded with random coil parameters (sources: - statistical analysis from unfolded peptides - random coil models (e.g. polymer model, Model-free analysis, Flory etc.) - unfolded with folded state parameters - different degrees of unfolded states • Chemical shifts • Relaxation rates • Heteronuclear NOE • Dipolar couplings • Scalar couplings Molecular Biophysics III: Spring 2006: Oznur Tastan

  21. 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 Residual structure in lysozyme WL-SME in urea WL-SME in water There are six clusters of residual structure in WL-SME. Klein-Seetharaman, 2002. Molecular Biophysics III: Spring 2006: Oznur Tastan

  22. Experiment: Mutation of W62 A single point mutation, W62G in cluster 3, disrupts all clusters in reduced and methylated lysozyme. Klein-Seetharaman, 2002. Molecular Biophysics III: Spring 2006: Oznur Tastan

  23. The Molten Globule(MG)State • Molten globule is characterized by • Absence of specific tertiary contacts • presence of some secondary structure • Native-like compactness • Presence of hydrophobic core Example: a-lactalbumin Molten globule observed in low pH Molecular Biophysics III: Spring 2006: Oznur Tastan

  24. The Molten Globule(MG)State • Absence of specific tertiary contacts • Presence of some secondary structure • Native-like compactness • Presence of hydrophobic core native pH5.4 MG-state pH2 unfolded state (in 9M urea,pH2) Kuwajima, K. 1989. Molecular Biophysics III: Spring 2006: Oznur Tastan

  25. The Molten Globule(MG)State • Absence of specific tertiary contacts • Presence of some secondary structure • Native-like compactness • Presence of loosely packed hydrophobic core. Katoka, 1997. Molecular Biophysics III: Spring 2006: Oznur Tastan

  26. The Molten GlobuleSignificance for Protein Folding Mechanism Disordered polypeptide collapse into the molten globule. According to one view, http://www.bmb.psu.edu/courses/bmb401H/Chapter7and8.pdf Molecular Biophysics III: Spring 2006: Oznur Tastan

  27. How do small single-domain proteins fold? • 20 small proteins(< 100 aa) are showed to fold : • simple two-state folding kinetics • show variation in their folding rates(microseconds to seconds) • all structures has to pass the transition state in order to reach the native state Dobson, 2003 Molecular Biophysics III: Spring 2006: Oznur Tastan

  28. Kinetics of Two-State Folding Chevron Plot of CI2 ln λ kfold kunfold [GdnHCl] ΔGŦ-D = GŦ - G°D = - RTlnkfold ΔGN-D = G°N – GŦ = - RTlnkunfold λ= kfold + kunfold An indicator of 2-state kinetics. Molecular Biophysics III: Spring 2006: Oznur Tastan

  29. Ф-value AnalysisCharacterization of the Transition State(TS) TS cannot be isolated or studied directly. Systematically introduce mutations in the native protein. Infer structure of TS from the energetics of the folded state (mutant versus wild-type). Ф=1: site of mutation is native-like in TS. Ф=0: site of mutation is unfolded in TS. Fractional Ф value: partial structure in TS. Ф = ΔΔGŦ-D / ΔΔGN-D Reproduced form Molecular Biophysics III: Spring 2006: Oznur Tastan

  30. Transition State Analysis:Case Study I: Chymotrypsin inhibitor(CI2) In the transition state of CI2 three residues with Ф-values >0.5 come together :A16,L49,I57. A hydrophobic core supporting the nucleation-condensation mechanism Molecular Biophysics III: Spring 2006: Oznur Tastan

  31. Complex pathwaysCase study II: hen lysozyme Most proteins (>100 aa) fold with observable intermediates. Thus cannot be approximated with simple 2-state kinetics. lysozyme Dill & Chan et al.1997 Molecular Biophysics III: Spring 2006: Oznur Tastan

  32. Understanding how lysozyme folds HX + NMR β α α α α β α α β α β Alpha and beta domains are two distinct folding units. Radford,et.al,1992 Molecular Biophysics III: Spring 2006: Oznur Tastan

  33. The details? HX labeling & EMS α domain is structured independently of the β domain in the early stages of folding. Near-UV CD unprotected α β Far-UV CD Large secondary structure is formed within the milliseconds of folding. Single exponential, the α domain forms before the near UV develops. The tertiary contacts are not fixed yet. Dobson,et.al. 1994 Molecular Biophysics III: Spring 2006: Oznur Tastan

  34. Intrinsic Trp flouresence A change in the some or all of the Trp occurs in early collapse in later intermediates and on formation of the native structure. Quenching of flouresence by iodine Exclusion of water in the early stages of folding Binding of ANS Maximal emission in the early stages. A relatively loosely packed, condensed state exists in the early stages of reaction. Dobson,et.al. 1994 Molecular Biophysics III: Spring 2006: Oznur Tastan

  35. Folding pathway of lysozyme Major >30% Minor ~10% Very rapidly alpha domain forms. Hydrophobic interior develops. Few tertiary interactions. Protective structure evolves. Dynamic and fluctuating beta domain. Alpha and beta domains are stabilized Dobson,et.al. 1994 Molecular Biophysics III: Spring 2006: Oznur Tastan

  36. Complementary approaches are essential! Dobson, 1998. Molecular Biophysics III: Spring 2006: Oznur Tastan

  37. Summary No clear unifying view of protein folding has yet emerged. Molecular Biophysics III: Spring 2006: Oznur Tastan

  38. Membrane Protein FoldingModel systems The lipid environment α-helical bundles β-barrels Mammalian Rhodopsin Bacteriorhodopsin OmpA Molecular Biophysics III: Spring 2006: Oznur Tastan

  39. Denaturation of bacteriorhodopsin Effects of Urea and Guanidinium Hydrochloride: almost none, not even on tertiary structure. Molecular Biophysics III: Spring 2006: Oznur Tastan

  40. Denaturation of Bacteriorhodopsin Formic acid Native SDS Secondary structure remains even in SDS. Molecular Biophysics III: Spring 2006: Oznur Tastan

  41. Why is it so difficult to disrupt secondary structure in membrane proteins? Molecular Biophysics III: Spring 2006: Oznur Tastan

  42. Thermodynamic considerations The engaging of polar backbone in H-bonds is favorable. White & Wimley,1999. Molecular Biophysics III: Spring 2006: Oznur Tastan

  43. Refolding of Bacteriorhodopsin in the lipid bilayers Refolded: fragments have near-identical helix content. C-1 C-2 1. C-1 and C-2 in SDS 2. C-1 and C-2 +lipid 3. Retinal omitted. Retinal reconstituted: %90 of the activity regenerated. BR can assemble in to the native structure when helices are inserted into the membrane independently. Popot, 1987. Molecular Biophysics III: Spring 2006: Oznur Tastan

  44. The two-stage hypothesis: Independent helix intermediate 1st Stage: Helix folds Independently 2nd Stage: Final packing and interactions between the helices are formed. New 3rd Stage: Biding of prosthetic groups, folding of loops, oligomerization… Two stage hypothesis may not hold in all cases. Engelman &Popot, 1990. Engelman & Popot, 2003. Figures from Klein-Seetharaman,2005. Molecular Biophysics III: Spring 2006: Oznur Tastan

  45. HR unfolded with AFM Helix G unfolded in two steps Part of helix E Short cytoplasmic segment HelixF Helix E unfoldswith helix D Helix C Helix A unfolds in two steps B-C loop Helix B Cooperative unfolding barriers are observed, in conflict with the 2-stage hypothesis. Cisneros, 2005. Molecular Biophysics III: Spring 2006: Oznur Tastan

  46. Folding Core Prediction of Rhodopsin Gaussian Network Model (GNM) FIRST Agreement with the mutational data (>%90 ). Folding core lies in the EC-TM domain interface. Evidence for the significance of long-range interactions in the folding of rhodopsin. Rader. et.al 2004 Molecular Biophysics III: Spring 2006: Oznur Tastan

  47. New Model: Long Range Interaction intermediate Klein-Seetharaman,2005. Molecular Biophysics III: Spring 2006: Oznur Tastan

  48. References • Anfinsen, C.B. (1973) "Principles that govern the folding of protein chains." Science 181 223-230. • Cisneros, D.A., D. Oesterhelt, and D.J. Muller, Probing origins of molecular interactions stabilizing the membrane proteins halorhodopsin and bacteriorhodopsin. Structure, 2005. 13(2): p. 235-42. • Dobson, C.M.Sali A., and Karplus, M., "Protein Folding: A Perspective from Theory and Experiment", Angew. Chem. Int. Ed. Eng. 37, 868-893 (1998). • Dobson, C.M. "Protein Folding and Misfolding", Nature 426, 884-890 ( 2003). • Dobson, C.M., P.A. Evans, and S.E. Radford, Understanding how proteins fold: the lysozyme story so far. Trends Biochem Sci, 1994. 19(1): p. 31-7. • Engelman, D. M., Chen, Y., Chin, C. N., Curran, R., Dixon, A. M., Dupuy, A, Lee, A., Lehnert, U., Matthews, E., Reshetnyak, Y., Senes, A., Popot, J-L. “Membrane Protein Folding: Beyond the Two Stage Model” FEBS Lett. (2003) 555:122-5. • Flory, P. J. (1969) Statistical Mechanics of Chain Molecules (Wiley, New York). • Fitzkee, N.C. and Rose, G.D. (2004). Reassessing random-coil statistics in unfolded proteins. Proc. Natl. Acad. Sci. 101: 12497–12502. • Klein-Seetharaman, J., Oikawa, M.,Wirmer, J., Duchardt, E., Ueda, T., Imoto, T., Smith, L.J., Dobson, C. and Schwalbe, H. (2002) Long-Range Interactions within a Non-Native Protein. Science 295, 1719-1722. • Klein-Seetharaman, J. (2005) Dual role of interactions between membranous and soluble portions of helical membrane receptors for folding and signaling. Trends in Pharmacological Science 26(4), 183-189 • Kuwajima, K. (1989). The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins: Struct. Funct. Genet. 6: 87–103. • Kataoka, M., Y. Hagihara, K. Mihara, and Y. Goto. (1993). Molten globule of cytochrome c studied by the small angle X-ray scattering. J. Mol. Biol. 229:591-596. • Radford, S. E., Dobson, C. M. & Evans, P. A. The folding of hen lysozyme involves partially structured • intermediates and multiple pathways. Nature 358, 302-307 (1992). • Pappu, R. V. , Srinivasan, R. & Rose, G. D. (2000) Proc. Natl. Acad. Sci. USA 97, 12565-12570. • Popot, J-L and Engelman D.M."Membrane Protein Folding and Oligomerization: The Two-Stage Model“ Biochemistry (1990), 29 (17), 4031-7. • Popot J.L., Gerchman S.E., Engelman D.M. (1987) Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically controlled two-stage process. J. Mol. Biol. 198:655-76 • Shortle D, Ackerman MS. (2001) Persistence of native-like topology in a denatured protein in 8 M urea. Science. Jul 20;293(5529):487-9. • White S. H. and Wimley, W. C. (1999).  Membrane protein folding and stability: Physical principles.  Annu. Rev. Biophys. Biomol. Struct. 28:319-365.  • http://www.otago.ac.nz/humannutrition/dietetics/gfx/philosophy.jpg, March 22, 2006. • http://www-nmr.cabm.rutgers.edu/academics/biochem694/2006BioChem412/Biochem.412_2-24-2006lecture.pdf, March 22, 2006. • http://www.makro.ch.tum.de/users/BFHZ/Scheibel/Scheibel%202003%20Bordeaux-1.pdf, March 22, 2006. Molecular Biophysics III: Spring 2006: Oznur Tastan

  49. Acknowledgements Dr. Judith Klein-Seetharaman Dr. Sanford Leuba & The class of MB3 (Spring 2006) Molecular Biophysics III: Spring 2006: Oznur Tastan

  50. QUESTIONS? http://www.otago.ac.nz/humannutrition/dietetics/gfx/philosophy.jpg Molecular Biophysics III: Spring 2006: Oznur Tastan

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