FOLDING KINETICS OF VILLIN 14T, A PROTEIN DOMAIN WITH A CENTRAL BETA-SHEET AND 2 HYDROPHOBIC CORES

The thermodynamics and kinetics of folding are characterized for villi n 14T, a 126-residue protein domain. Equilibrium fluorescence measurem ents reveal that villin 14T unfolds and refolds reversibly. The foldin g kinetics was monitored using stopped-flow with fluorescence and quen ched-flow with NMR and mass spectrometry. Unfolding occurs in a single -exponential phase in the stepped-flow experiments, and about 75% of t he total amplitude is recovered in the fast phase of refolding. The re maining 25% of the amplitude probably represents trapping in cis-trans proline isomerization pathways. At 25 degrees C, the stability estima te obtained by extrapolation from the transition region of the stopped -flow chevron matches the stability value from equilibrium urea titrat ions (Delta G = 9.7 kcal/mol, Nz value = 2.2 kcal mol(-1) M-1). At low final urea concentrations, however, the refolding kinetics deviates f rom the two-state model, indicating the formation of an intermediate. Under these conditions, quenched-flow followed by NMR and mass spectro metry show no detectable hydrogen-bonded intermediate in the fast refo lding phase. In contrast, agreement is observed between the equilibriu m and kinetic estimates of stability at 37 degrees C (Delta G = 6.0 kc al/mol, m value = 1.6 kcal mol(-1) M-1), at all observed urea concentr ations, demonstrating apparent two-state folding at this temperature. This result shows that the two-state folding model, previously applied to small domains with single, central hydrophobic cores, can also des cribe the folding of a larger domain with multiple core structures.