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. Kinetic models are used to fit and interpret the measured data. We have developed a novel method for measuring the kinetics of loop formation in peptides in which one position is labeled with tryptophan and the other with cysteine or cystine. 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 quantitatively characterized this method for peptides having the sequence cys-(ala-gly-gln)j-trp. For these peptides, the diffusion-limited rate, which is the rate of contact formation, can be obtained from measurements of the temperature- and viscosity-dependence of the quenching rates. These experiments also provide values for the reaction-limited quenching rates, which provide information on the equilibrium end-to-end distribution. The dependence of the reaction-limited rates on peptide length can be accurately calculated using the distance-dependence of the quenching rate of the tryptophan triplet by cysteine obtained by embedding the free amino acids in a trehalose glass at room temperature, and a one-dimensional potential of mean force obtained from the end-to-end distance distribution for a worm-like chain. The apparent turnover in the reaction-limited rate at short chain lengths can be attributed to the stiffness of a chain having a persistence length of 0.65 nm. The diffusion-limited rates can be calculated by treating the dynamics as diffusion on the same one-dimensional potential of mean force. The diffusion coefficient for the chain ends required to fit the diffusion-limited rates is about 1.7(10^-6) cm^2s-1 at a viscosity of 1 cp, approximately 10 times smaller than the value expected for free diffusion of the contacting residues. We have recently focused on using this technique to understand the dynamics of unfolded and partially-folded polypeptides. To explore the effects of excluded volume we have added a 9-residue 'tail' at each end of the 11-residue peptide. In 6 M guanidine hydrochloride, the peptide expands, the rates for slow down and the length-dependence of the reaction-limited rates becomes larger for peptides longer than 10 amino acids. The rates for the peptides with extended tails are about a factor of 2 slower than that for the 11-mer. Simple extensions of the wormlike chain model that include excluded volume produce similar changes in the reaction-limited rates. We have also begun to characterize the loop dynamics for a mutant of the cold shock protein from the hyperthermophylic bacterium Thermotoga maritime, CspTm. In 6 M guanidine hydrochloride the reaction-limited rates for a 28-residue loop excised from this protein are about 2-fold slower than those for our (ala-gly-gln)j reference peptide. This suggests that this sequence is stiffer. When the intact protein is compared with the 28-residue fragment, the rates are slower, as expected from excluded volume effects. We are also beginning to investigate the behavior of these peptides at lower guanidine concentrations

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
United States
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