| Summary: | The problem of protein dynamics is introduced and its significance explained. Properties of the oxygen-storage protein myoglobin (Mb) as a model system for dynamics studies are discussed. Special attention is paid to Mb's physiological role, and the basic quantities that describe the protein's function. Background on ligand binding experiments in Mb is reviewed and appropriate mathematical models established. The specific goal of this work is to determine as many as possible of the model parameters (pre-exponentials and activation enthalpies of intrinsic rate coefficients), with a view towards calculation of one functionally important quantity, the CO affinity at physiological temperatures. Relaxation spectroscopy is a powerful means for studying dynamics. Different perturbations and observables are considered, and two kinetic methods are introduced: temperature-derivative spectroscopy (TDS), a non-isothermal technique that measures the derivative of a population with respect to temperature; and deep-level transient spectroscopy (DLTS), an isothermal technique that determines the behavior of a small range of rate coefficients as a function of temperature. These "rate-window" methods are shown to be widely applicable and may prove highly advantageous in difficult measurements such as kinetic X-ray crystallography. TDS and DLTS were used to study the rebinding of CO to sperm whale Mb after photolysis. FTIR measurements of geminate rebinding in the CO-stretch bands show distributed activation enthalpies with different distributions for each band, crossing between two bands that correspond to photolyzed ligands, and kinetic hole-burning. The distributions of activation enthalpies are well described by gaussians; the results match and complement those of traditional multi-rate methods. Further experiments determined the barriers to entry to and escape from the heme pocket for two of the bands. Information about barriers to different kinds of conformational changes were also obtained. The kinetic differences among different protein conformations provide a mechanism by which the affinity of Mb might be modified in response to physiological demands. It is shown that this effect could be larger than that of the R- to T-state change in hemoglobin. Findings from the physiological and biochemical literature consistent with this possibility are pointed out, and specific tests are proposed.
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