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Transition States in Protein Folding

Transition States in Protein Folding. Max Planck Institute of Colloids and Interfaces Department of Theory and Bio-Systems. Thomas Weikl. Mutational -value analysis of the folding kinetics Modeling -values for -helices Modeling -values for small -sheet proteins.

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Transition States in Protein Folding

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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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