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CS 9: f ,m analysis I

CS 9: f ,m analysis I. Biochemistry 655 31 January 2011. Goals. Introduce the style and substance of Alan Fersht’s work “ … do something with physical chemistry that the crystallographers could not do .” Introduce the physical chemistry of (protein folding) mechanisms.

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CS 9: f ,m analysis I

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  1. CS 9: f,m analysis I Biochemistry 655 31 January 2011

  2. Goals • Introduce the style and substance of Alan Fersht’s work “… do something with physical chemistry that the crystallographers could not do.” • Introduce the physical chemistry of (protein folding) mechanisms. What is meant by “transition state”? How do point mutants divide up space How do rate measurements divide up time? How does combining behavior in space and time give us mechanism? • Distinguish between equilibrium and rate constants: Keq = kf/kb • Review the relationships between these constants and the corresponding free energies: • DGKeq = -RT*ln(Keq); • DG‡ = -RT*ln(kf,b) • Define and interpret m, f. m is the urea-dependence of an equilibrium or rate constant (Tanford). f is related to the ratio of rates to the corresponding equilibria (Fersht). • Fersht’s mutational f,m analysis was a monumental breakthrough. Characterized transition states and intermediates for the first time. Essentially all folding research since has been predicated on the analysis he pioneered.

  3. Understanding mechanism(…”what crystallographers could never do!”) • Overall strategy has three parts (I call these “Fersht’s rules”): • Characterize states along the path • Show that they form fast enough to be on the path. • Show that they react fast enough to be on the path

  4. Matouschek’s work: an experimental coup providing evidence for a folding mechanism!

  5. Chemical denaturation - Tanford • Urea denatures by dissolving tertiary structure. • Tanford (~1960) showed that protein folding equilibria depended linearly on [urea]. • He argued that the slope of this dependence, m, was a measure of the increase in surface area in the denatured state.

  6. m had been around since the early 60’s

  7. Point mutants localize spatial effects

  8. Rapid kinetics localizes effects in time 0.64 M Urea

  9. f was a radical innovation, bridging space and time

  10. 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, 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 be used to detect intermediates!!

  11. Detecting Intermediates 0.64 M Urea

  12. Thermodynamic analysis of mutant behavior…

  13. Folding models: now much more specific

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

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