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CS 10: Sequential vs parallel paths

CS 10: Sequential vs parallel paths. Biochemistry 655 2 February 2011. Goals. Review the use of refolding kinetics to identify (molten globule) intermediates: unifying the minimal Barnase, CI2 folding schemes (Otzen). A more detailed look at the folding transition, D => I (Dalby).

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CS 10: Sequential vs parallel paths

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  1. CS 10: Sequential vs parallel paths Biochemistry 655 2 February 2011

  2. Goals • Review the use of refolding kinetics to identify (molten globule) intermediates: unifying the minimal Barnase, CI2 folding schemes (Otzen). • A more detailed look at the folding transition, D => I (Dalby). • 1st order vs 2nd order transitions • Cooperativity • Detecting parallel paths experimentally (Fersht). • Linear free energy relations • The Brønsted relation • Compare experimental and computationally-derived f values (Dagget).

  3. Relating equilibria and rates • An equilibrium constant can be expressed as the ratio of forward and reverse rate constants: • Keq,u = ku/kf = 1/Keq,f • This provides predicted folding rates,kf,pred, once equilibrium and unfolding rate constants are known: • kf = ku/Keq,u = Keq,f * ku • kf,pred = ku/Keq,u ≠ kf,obs • => Refolding experiments can detect intermediates!!

  4. Detecting Intermediates

  5. Thermodynamic analysis of mutant behavior…

  6. Space Time Time Time Time …characterizes in space and time

  7. Folding models: now much more specific

  8. (Variable 2-state) First-order; coooperative The minimal barnase folding scheme

  9. Chymotrypsin Inhibitor 2 (Otzen)

  10. A Unified model? >1 Module

  11. Detection of parallel pathways-1 core surface

  12. Detection of parallel pathways-2 Major a-helix in Barnase

  13. A unified model?

  14. The folding transition D => I • First order transitions imply that Keq changes under different conditions. • kf is a first-order transition, a single exponential • This behavior is also called “cooperative”. • Cooperativity is a matter of degree (ie., there is always fine structure at some level of detail). • Second-order transitions imply that structures change under different conditions. • Different conditions produce different species. • A single species is present at any set of conditions. • This model involves progressive conversion of D => I. • Phi and m value analysis can help distinguish these possibilities.

  15. Folding behavior of Barnase mutants

  16. Phase Diagram Analysis

  17. Lots of experiments!!!

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