Time-resolved absorption and fluorescence spectroscopy are used to study the dynamics of protein structural changes subsequent to rapid mixing or excitation with short laser pulses. Molecular models for the protein dynamics are used to fit and interpret the measured data. A. We have begun to develop new microtechniques for producing rapid mixing and temperature jumps. In collaboration with James Knight and Robert Austin of Princeton University, we have been exploring the use of microdevices fabricated using integrated circuit technology to produce rapid mixing devices. We have also begun to develop a micro-T-jump aparatus which will utilize continuous illumination from an infrared laser to heat small volumes (100 :m3) of water in thin layers. The rapid thermal equilibration of the thin layer will permit both positive and negative temperature jumps to be initiated on the microsecond time scale. B. Laser temperature jump studies have been carried out on a 21-residue alanine peptide containing tryptophan (W) at position 1 and histidine (H) at position 5. The interaction between the charged H quenches the fluorescence of W in the helical state. We observe that melting of the helix is biphasic, with rates of about 3(10/6) s-1 and 4(10/7) s-1 at 25 C. About 80% of the amplitude is observed for the slow relaxation. We have developed a 'kinetic zipper' model which correctly predicts the relative time scales and amplitudes. The faster relaxation results from 'unzipping' on helix ends, while the slower relaxation results from helical peptides crossing the nucleation free energy barrier. C. We have studied the folding of an isolated $ hairpin from the C-terminus of protein GB1. Folding is followed by monitoring the fluorescence of a single tryptophan residue in the fragment. Apparent 2-state behavior is observed with a single relaxation rate of 2(10/5) s-1 at 25 C, about 20 times slower than formation of the """""""" helix. We have developed a simple statistical mechanical model to explain these results, which shows that the difference in rate results from both the limited number of positions in which the turn can successfully initiate and the fact that the hairpin must be almost completely formed before it becomes stable.
