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Sequential vs. Parallel Paths in Biochemical Folding: Analyzing Refolding Kinetics and Intermediates

This review explores the role of refolding kinetics in identifying molten globule intermediates, unifying folding schemes of Barnase and CI2. It examines first-order versus second-order transitions, cooperativity, and experimental detection of parallel folding pathways. Key concepts include linear free energy relations and the Brønsted relation, with discussions on equilibrium constants and their implications on predicted folding rates. The analysis also considers the folding behavior of Barnase mutants and the significance of experimental and computational approaches in characterizing folding dynamics.

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Sequential vs. Parallel Paths in Biochemical Folding: Analyzing Refolding Kinetics and Intermediates

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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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