Temperature dependence of the folding and unfolding kinetics of the GCN4 leucine zipper via C-13(alpha)-NMR

Studies by one-dimensional NMR are reported on the interconversion of folde d and unfolded forms of the GCN4 leucine zipper in neutral saline buffer. T he peptide bears 99% C-13(alpha) labels at three sites: V9, L12, and G31. T ime-domain C-13(alpha)-NMR spectra are interpreted by global Bayesian lines hape analysis to extract the rate constants for both unfolding and folding as functions of temperature in the range 47-71 degreesC. The data are well fit by the assumption that the same rate constants apply at each labeled si te, confirming that only two conformational states need be considered. Resu lts show that 1) both processes require a free energy of activation; 2) unf olding is kinetically enthalpy-opposed and entropy-driven, while folding is the opposite; and 3) the transition state dimer ensemble averages similar to 40% helical. The activation parameters for unfolding, derived from NMR d ata at the elevated temperatures where both conformations are populated, le ad to estimates of the rate constant at low temperatures (5-15 degreesC) th at agree with extant values determined by stopped-flow CD via dilution from denaturing media. However, the corresponding estimated values for the fold ing rate constant are larger by two to three orders of magnitude than those obtained by stopped flow. We propose that this apparent disagreement is ca used by the necessity, in the stopped-flow experiment, for initiation of ne w helices as the highly denaturant-unfolded molecule adjusts to the newly c reated benign solvent conditions. This must reduce the success rare of coll isions in producing the folded molecule. In the NMR determinations, however , the unfolded chains always have a small, but essential, helix content tha t makes such initiation unnecessary. Support for this hypothesis is adduced from recent extant experiments on the helix-coil transition in single-chai n helical peptides and from demonstration that the folding rate constants f or coiled coils, as obtained by stopped flow, are influenced by the nature of the denaturant used.