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Unraveling Protein Folding Kinetics: Transition States Modeling Analysis

Explore the complex kinetics of protein folding, focusing on transition states and mutational analysis. Understand how proteins fold into their native structures and the role of intermediates. Utilize modeling to study α-helices and small β-sheet proteins.

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Unraveling Protein Folding Kinetics: Transition States Modeling Analysis

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  1. Transition States in Protein Folding Max Planck Institute of Colloids and Interfaces Department of Theory and Bio-Systems Thomas Weikl

  2. Mutational -value analysis of the folding kinetics Modeling -values for -helices Modeling -values for small -sheet proteins Overview

  3. The kinetics problem: How does a protein fold into its structure? Protein folding problems • The structure problem: In which native structure does a given sequence fold?

  4. The ”new view”: Many small proteins fold without detectable intermediates (2-state proteins) How does a protein fold? • The Levinthal paradox: How does a protein find its folded conformation as ”needle in the haystack“? • The ”old view”: Metastable folding intermediates guide a protein into its native structure

  5. Donor and acceptor dyes at chain ends • State-dependent transfer efficiency 2-state folding: Single molecules Schuler et al., Nature 2002

  6. rapid mixing to initiate folding spectroscopic signal denatured state D native state N • single-exponential relaxa-tion for 2-state process: 200 0 100 300 time (ms) 2-state folding: Protein ensemble N protein + den. H20

  7. T G D N • Mutations change the folding ratek and stability GN–D T’ G • Central quantities: -values T D N’ GT–D N   GN–D Mutational analysis of 2-state folding • Transition state theory: k exp(-GT–D)

  8. T’ G T D N’ N  = 0: mutated residue is unstructured in T Traditional interpretation of  T’ G T D N’ N = 1: mutated residue is native-like structured in T

  9. Inconsistencies: - some ’s are <0 or >1 - different mutations of the same residue can have different -values Goldenberg, NSB 1999 -values Traditional interpretation of  • : degree of structure formation of a residue in T

  10. Our finding:  Example: -helix of CI2 mutation  • -values for mutations in the helix range from -0.25 to 1.06 S12G S12A E15D E15N A16G K17G K18G I20V L21A L21G D23A K24G 0.29 0.43 0.22 0.53 1.06 0.38 0.70 0.40 0.25 0.35 -0.25 0.10 Itzhaki, Otzen, Fersht,1995

  11. Mutational -value analysis of the folding kinetics Modeling -values for -helices Modeling -values for small -sheet proteins

  12. Helix cooperativity • we assume that a helix is either fully formed or not formed in transition-state conformation Ti • we have two structural parameters per helix: - the degree ofsecondary structure  in T - the degree of tertiary structure t in T

  13. T G D N • general form of -values for mutations in an -helix: Splitting up free energies • we split up mutation-induced free energy changes into secondary and tertiary components:

  14.   1.0 t  0.15 D23A -values for-helix of CI2 mutational data for CI2 helix: general formula: 

  15.   1.0 t  0.45 -values for helix 2 of protein A mutational data for helix 2: general formula:  3 1 2

  16. Summary Consistent interpretation of -values for helices: • with two structural parameters: the degrees of secondary and tertiary structure formation in T • by splitting up mutation-induced free energychanges into secondary and tertiary components C Merlo, KA Dill, TR Weikl, PNAS 2005 TR Weikl, KA Dill, JMB 2007

  17. Mutational -value analysis of the folding kinetics Modeling -values for -helices Modeling -values for small -sheet proteins

  18. Modeling 3-stranded -proteins • WW domains are 3-stranded -proteins with two -hairpins • we assume that each hairpin is fully formed or not formed in the transition state

  19. Evidence for hairpin cooperativity • 3sis a designed 3-stranded-protein with 20 residues • transition state rigorously determined from folding-unfolding MD simulations • result: either hairpin 1 or hairpin 2 structured in T Rao, Settanni, Guarnera, Caflisch, JCP 2005

  20. the folding rate is: • -values have the general form: A simple model for WW domains • we have two transition-state conformations with a single hairpin formed

  21. exp -values for FBP WW domain • a first test: ’s for mutations affecting only hairpin 1 should have value 1 • general formula:

  22. single-parameter fit: 1 0.77 2= 1- 1  0.23 exp theo -values for FBP WW domain • general formula:

  23. Summary Reconstruction of transition states from mutational -values based on: • substructural cooperativity of helices and hairpins • splitting up mutation-induced free energychanges C Merlo, KA Dill, TR Weikl, PNAS 2005 TR Weikl, KA Dill, J Mol Biol 2007 TR Weikl, Biophys J 2008

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