Thermodynamics and kinetics of a folded-folded ' transition at valine-9 ofa GCN4-like leucine zipper

Spin inversion transfer (SIT) NMR experiments are reported probing the ther modynamics and kinetics of interconversion of two folded forms of a GCN4-li ke leucine zipper near room temperature. The peptide is C-13(alpha)-labeled at position V9(a) and results are compared with prior findings for positio n L13(e). The SIT data are interpreted via a Bayesian analysis, yielding lo cal values of T-1a, T-1b, k(ab), k(ba), and K-eq as functions of temperatur e for the transition F-a(V9) reversible arrow F-b(V9) between locally folde d dimeric forms. Equilibrium constants, determined from relative spin count s at spin equilibrium, agree well with the ratios k(ab)/k(ba) from the dyna mic SIT experiments. Thermodynamic and kinetic parameters are similar for V 9(a) and L13(e), but not the same, confirming that the molecular conformati onal population is not two-state. The energetic parameters determined for b oth sites are examined, yielding conclusions that apply to both and are rob ust to uncertainties in the preexponential factor (kT/h) of the Eyring equa tion. These conclusions are 1) the activation free energy is substantial, r equiring a sparsely populated transition state; 2) the transition state's e nthalpy far exceeds that of either F-a or F-b; 3) the transition state's en tropy far exceeds that of F-a, but is comparable to that of F-b; 4) "Arrhen ius kinetics" characterize the temperature dependence of both k(ab) and k(b a), indicating that the temperatures of slow interconversion are not below that of the glass transition. Any postulated free energy surface for these coiled coils must satisfy these constraints.