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Important information on the mechanism and dynamics of a molecular process can be obtained by studying the dependence of its kinetics on solvent viscosity. Classic examples are the investigation of the role of diffusion in determining the rate of a bimolecular reaction (1) and the influence of solvent friction on a unimolecular reaction rate (2). Other examples include the effect of solvent viscosity on the motion of small molecules inside proteins (3) and the contribution of diffusive processes to the rate-limiting steps in protein folding (4). Here we show how such studies can be used to gain insight into the dynamics of conformational changes in proteins.
The carbon monoxide complex of myoglobin (MbCO) is dissociated by light. The conformation of the photoproduct is unstable, and the protein relaxes to the conformation of the unliganded molecule by a small but global displacement of protein atoms on one side of the heme. We have investigated the kinetics of this conformational change as a function of solvent viscosity by using high-precision, time-resolved absorption measurements after photodissociation by nanosecond laser pulses. A representative set of time-resolved absorption spectra in a 79% by weight solution of glycerol in water at 20 deg C is shown in Fig. 1. (Figure omitted) The time course of the decrease in the overall amplitude of the difference spectra measures the ligand rebinding kinetics, which take place in two phases (Fig. 2A). (Figure omitted) The first, nonexponential phase, with a half-time of about 200 ns, corresponds to geminate rebinding, that is, unimolecular rebinding of CO to the heme from which it was photodissociated (5). The second phase, at about 1 ms, corresponds to bimolecular rebinding of CO from the solvent. There are also changes in the shape of the spectra, which are primarily spectral changes of the deoxyheme photoproduct (Fig. 2B). (Figure omitted) The corresponding amplitudes (Fig. 2, B and C) monitor the extent of the deviations of the observed spectra from the average spectrum shown in Fig. 2A. (Figures omitted). We interpret these spectral changes as arising from protein conformational changes following photodissociation. Lambright et al. (6) have independently observed these kinetics but did not investigate their viscosity dependence.
The similarity of the deoxyheme spectral changes to those observed for hemoglobin suggests that they...