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 kinetic models are used to fit and interpret the measured data. (A) Laser temperature jumps have been used to study the folding of a peptides which form alpha helices and beta hairpins. The helix-forming peptide is a21-residue alanine peptide containing tryptophan (W) at position 1 and histidine(H+) at position 5. In this peptide the charged H+ quenches the fluorescence of Win the helical state. Most of the fluorescence change occurs in a single 220 ns relaxation at the midpoint of the folding transition (300 K). The hairpin-forming peptide is a 16-residue fragment from the C-terminus of protein GB1. Folding is followed by monitoring the fluorescence of a single tryptophan residue which is partially buried in the folded state. Apparent 2-state behavior is observed with a single relaxation rate of 2(10^5)/s at 25 C. To obtain accurate values for the activation barriers for these processes, we have measured the dependence of the kinetics of these systems on temperature and solution viscosity using the sugars sucrose and glucose as viscogenic agents. When analyzed using a power- law viscosity dependence, we find the kinetics of both systems are damped by the solvent. The helix-coil kinetics exhibit a viscosity exponent of about 0.6 and the hairpin-coil kinetics by an exponent of about 1.0. When solvent damping is included, the hairpin-coil kinetics can be explained using only the equilibrium contributions to the free energy barrier while the temperature dependence of the helix-coil kinetics is significantly larger than that predicted by the equilibriu mbarrier described by our model, suggesting that there may be a significant enthalpic contribution to nucleation of helices which is not included inequilibrium descriptions of helix formation. (B) We have developed a simple statistical model to describe the folding kinetics in terms of two states for each peptide bond, native and random. This model can be used to predict how folding rates depend on the peptide sequence, particularly for helix formation, where the contribution of individual amino acids to the helix stability has been extensively studied. The model predicts that when the helix is destabilized by introducing residues with decreased helix propensities, the folding rates will decrease and the unfolding rates will not change. If these residues are positioned to produce stabilizing interactions in the folded helix, both rates will decrease. We have begun to test these predictions in peptides containing leucine and isoleucine. (C) We have developed a generic method for measuring the kinetics of loop formation in peptides in which one position is labelled with tryptophan and the another with a cysteine or cystine. The trypophan, excited to its lowest triplet state by a 280-290 nm laser pulse, is efficiently quenched by the sulfur- containing residue upon loop formation. We have measured loop-fomation rates for a series of peptides composed of triplet repeats having the sequence ala-gly-gln. The rates decrease from about 3(10^7)/s for a 4-residue peptide to about 7(10^6)/s for a 19-residue peptide. When the peptide is stiff, as for proline peptides, the triplet state is unquenched. The triplet probe provides anew tool for studying short-range interactions in peptides and proteins.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Intramural Research (Z01)
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U.S. National Inst Diabetes/Digst/Kidney
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