Time-resolved absorption and fluorescence spectroscopy are usedto study the dynamics of protein structural changes subsequent torapid mixing or excitation with short laser pulses. Molecularkinetic models are used to fit and interpret the measured data. (A) Laser temperature jumps have been used to study the foldingof a 21-residue alanine peptide containing tryptophan (W) atposition 1 and histidine (H+) at position 5. In this peptide thecharged H+ quenches the fluorescence of W in the helical state. Most of the fluorescence change occurs in a single 220 nsrelaxation at the midpoint of the folding transition (300 K) withan apparent activation energy of 8 kcal/mol. We have alsostudied the folding of an isolated hairpin from the C-terminus ofprotein GB1. Folding is followed by monitoring the fluorescenceof a single tryptophan residue which is partially buried in thefolded state. Apparent 2-state behavior is observed with a singlerelaxation rate of 2(10^5)/s at 25 C and an apparent activationenergy of 9 kcal/mol. (B) We have measured the dependence of the kinetics ontemperature and solution viscosity for these systems using thesugars sucrose and glucose as viscogenic agents. When analyzedusing a power-law viscosity- dependence, we find the kinetics otboth systems are damped by the solvent. The helix-coil kineticscan be characterized by a viscosity exponent of about 0.6 and thehairpin-coil kinetics by an exponent of about 1.0. (C) We have used a simple statistical mechanical model toexplain these results. When solvent damping is included, thehairpin- coil kinetics can be explained using only the equilibriumcontributions to the free energy barrier. The temperaturedependence of the helix-coil kinetics is significantly largerthan that which arises from the equilibrium barrier described byour model, suggesting that there may be a significant enthalpiccontribution to nucleation of helices which is not included inequilibrium descriptions of helix formation. Munoz and Eatonhave found that this relatively simple model can be extended todescribe the folding kinetics of proteins. (D) We have developed a generic method for measuring thekinetics of loop formation usingpeptides in which one end islabelled with tryptophan and the other with a disulfide. Thetrypophan, excited to its lowest triplet state by a 280-290 nmlaser pulse, is efficiently quenched upon loop formation by thecystine residue. We have measured a rate of 2(10^7)/s for a10- residue peptide. - Time-resolved spectroscopy; Protein folding and dynamics
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