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 measured the kinetics of loop formation for peptides, and for both the cold shock protein from Thermotoga Maritima CspTm and the folding subdomain of villin. To measure loop formation, one position in the chain is labeled with tryptophan and another with 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 had previously characterized this method for peptides having the sequence cys-(ala-gly-gln)j-trp, where the diffusion- and reaction-limited rates were obtained from measurements of the temperature- and viscosity-dependence of the quenching rates. The diffusion-limited rate is the rate of contact formation, while the reaction-limited rate provides information on the equilibrium end-to-end distribution. The turnover in the reaction-limited rate at short chain lengths was attributed to the stiffness of a chain having a persistence length of 0.65 nm. We have recently focused on using this technique to understand the dynamics of unfolded proteins and 'swollen' polypeptides. In 6 M guanidine hydrochloride (GdmCl), the peptides expand, the rates for slow down and the length-dependence of the reaction-limited rates increases for peptides longer than 10 amino acids as expected for chains 'swollen' by excluded volume. To further explore the effects of excluded volume we have carried out flourescence resonance energy transfer (FRET) experiments and added 9-residue 'tails' at each end of an 11-residue peptide. The FRET results show that the average end-to-end distance of the 11-residue sequence dansyl-(ala-gly-gln)3-trp increases by about 15% in 6 M GdmCl. The rates for the peptides with extended tails are about a factor of 2 slower than that for the 11-mer. Simulations of wormlike chains that include excluded volume produce similar changes in the the end-to-end distances and the reaction-limited rates. Reaction-limited rates for a 37-residue loop between Trp29 and Cys67 in unfolded CspTm have been measured at 1.5 to 6 M GdmCl. The quenching kinetics are more complex than those of the peptides, exhibiting stretched-exponential decays, [exp(-kt)^B] with the exponent B ~ 0.7. The origin of this complexity is not yet clear, but suggests that the unfolded protein must contain subpopulations with lifetimes comparable to the observed quenching rates (10 microseconds). The measured rates increase by about a factor of 2 at the lowest GdmCl concentration, suggesting that there is a decrease in the average distance between the tryptophan and cysteine residues. This finding is consistent with recent single molecule FRET experiments by Schuler et al. (2002). At 6 M GdmCl the rate is about 2-fold slower than those for the (ala-gly-gln)j reference peptide, suggesting that the protein sequence is stiffer. The average rate for the intact protein is also slightly slower than that of the 40-residue fragment His28-Cys67. Somewhat similar results have been obtain for the villin subdomain which has a tryptophan in position 23 and an N-terminal cysteine. This sequence also exhibits stretched exponential quenching rates with B ~ 0.8, The measured rate at 6 M GdmCl is also about 10 microseconds, despite the fact that this loop is only 21 residues long. Since the rate would be expected to increase more than two-fold for chains of comparable stiffness, this result suggests that the villin sequence is either significantly stiffer or more structured than that of CspTm.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Intramural Research (Z01)
Project #
1Z01DK029032-05
Application #
6810204
Study Section
(LCP)
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2003
Total Cost
Indirect Cost
Name
U.S. National Inst Diabetes/Digst/Kidney
Department
Type
DUNS #
City
State
Country
United States
Zip Code
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