Nonequilibrium protein folding dynamics: laser-induced pH-jump studies of the helix-coil transition

The kinetics of the helix-coil transition of poly-l-glutamate were measured in the range of 40 ns to 10 s using a laser-induced pH-jump coupled with time-resolved infrared spectroscopy. Folding of the polypeptide in D2O was initiated by photolyzing o-nitrobenzaldehyde, which releases a deuteron, creating a rapid decrease in pD. Side-chain deuteration and conformational changes were monitored independently by varying the IR probe wavelength. The kinetics of the peptide conformational changes observed in the amide I region depended on the initial fraction of helical residues. With essentially no initial helix, amide I absorption changes were indistinguishable from those of instrument response, leading to the conclusion that helix initiation occurs in less than 40 ns. When the initial helix fraction is 0.13, the folding lifetime is lengthened to 625 ns, as predicted by helix-coil theory. We also observe evidence for a kinetically-trapped, nonproductive intermediate formed as the result of rapid deuteration of the unfolded state.