Nanosecond lasers were used to measure the rate of conformational changes in myoglobin after ligand dissociation at ambient temperatures. At low solvent viscosities the rate is independent of viscosity, but at high viscosities it depends on approximately the inverse first power of the viscosity. Kramers theory for unimolecular rate processes can be used to explain this result if the friction term is modified to include protein as well as solvent friction. The theory and experiment suggest that the dominant factor in markedly reducing the rate of conformational changes in myoglobin at low temperatures (less than 200 K) is the very high viscosity (greater than 10(7) centipoise) of the glycerol-water solvent. That is, at low temperatures conformational substates may not be "frozen" so much as "stuck."

Initiation of protein folding by light can dramaticafly improve the time resolution of kinetic studies. Here we present an example ofan optically triggered folding reaction by using nanosecond photodissociation of the heme-carbon monoxide complex of reduced cytochrome c. The optical trigger is based on the observation that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide to the covalently attached heme group in the unfolded state. Photodissociation of the carbon monoxide thus triggers the folding reaction. We used time-resolved absorption spectroscopy to monitor binding at the heme. Before folding begins we observe transient binding of both nonnative and native ligands from the unfolded polypeptide on a microsecond time scale. Kinetic modeling suggests that the intramolecular binding of methionine-65 and -80 is faster than that of histidine-26 and -33, even though the histidines are doser to the heme. This optical trigger should provide a powerful method for studying chain collapse and secondary structure formation in cytochrome c without any limitations in time resolution.Protein folding generally occurs in two phases, one rapid and one slow. The rapid phase is the collapse of the unfolded polypeptide into a compact structure and the formation of secondary structure. The slow phase is the subsequent, often multistep rearrangement to the native conformation (1-7). Because of the limited time resolution (milliseconds) of conventional stopped-flow mixing experiments, the complete time course of protein folding has not yet been observed. A dramatic improvement in time resolution would result if protein folding could be initiated by light.Here we present an example of an optically triggered folding reaction. We take advantage of the fact that under destabilizing conditions cytochrome c can be unfolded by preferential binding of carbon monoxide (CO) to the covalently attached heme group in the unfolded state. The folding reaction can thus be triggered by photodissociating the CO complex. In this study, we used time-resolved absorption spectroscopy with nanosecond lasers to monitor binding events at the heme (8-11). Although rebinding of CO prevents the complete formation of the native conformation, the rapid recovery of the sample permits repetitive photolysis and therefore the acquisition of high signal/noise transient spectra for investigating submillisecond events. Simulations of the multiwavelength data with kinetic models were carried out to generate the spectra of intermediates, as well as the rate constants connecting them. We find that before folding begins there is transient binding of both nonnative and native ligands from the unfolded polypeptide chain on a microsecond time scale. MATERIALS AND METHODSTye VI horse heart cytochrome c from Sigma was purified by ion-exchange chromatography using carboxymethyl-cellulose (Whatman) (12), reduced with sodium dithionite anaerobically, and separated anaerobically from the sodium dithionite by gel filtrat...

Absorption spectroscopy with nanosecond time resolution shows that myoglobin undergoes conformational relaxation on the same time scale as geminate rebinding of carbon monoxide. Ligand rebinding following photodissociation of the heme-CO complex was measured from the amplitude of the average difference spectrum, while conformational changes were measured from changes in the detailed shape of the Soret spectra of the deoxyhemes. Experiments in which the solvent viscosity was varied between 1 and 300 cP and the temperature between 268 and 308 K were analyzed by fitting the multiwavelength kinetic data with both empirical and molecular models. Novel numerical techniques were employed in fitting the data, including the use of singular value decomposition to remove the effects of temperature and solvent on the spectra and of a Monte Carlo method to overcome the multiple minimum problem in searching parameter space. The molecular model is the minimal model that incorporates all of the major features of myoglobin kinetics at ambient temperatures, including a fast and slow rebinding conformation and two geminate states for each conformation. The results of fitting the kinetic data with this model indicate that the geminate-rebinding rates for the two conformations differ by at least a factor of 100. The differences between the spectra of the two conformations generated from the fits are similar to the differences between those of the R and T conformations of hemoglobin. In modeling the data, the dependence of the rates on temperature and viscosity was parametrized using a modification of Kramers theory which includes the contributions of both protein and solvent to the friction. The rate of the transition from the fast to the slow rebinding conformation is found to be inversely proportional to the viscosity when the viscosity exceeds about 30 cP and nearly viscosity independent at low viscosity. The viscosity dependence at high viscosities suggests that the two conformations differ by the global displacement of protein atoms on the proximal side of the heme observed by X-ray crystallography. We suggest that the conformational change observed in our experiments corresponds to the final portion of the nonexponential conformational relaxation recently observed by Anfinrud and co-workers, which begins on a picosecond time scale. Furthermore, extrapolation of our data to temperatures near that of the solvent glass transition suggests that this conformational relaxation may very well be the one postulated by Frauenfelder and co-workers to explain the decrease in the rate of geminate rebinding with increasing temperature above 180 K.

We have analyzed the nanosecond-millisecond kinetics of ligand binding and conformational changes in hemoglobin. The kinetics were determined from measurements of precise time-resolved optical spectra following nanosecond photodissociation of the heme-carbon monoxide complex. To fit the data, it was necessary to extend the two-state allosteric model of Monod, Wyman, and Changeux (MWC) to include geminate ligand rebinding and nonexponential tertiary relaxation within the R quaternary structure. Considerable simplification of the model is obtained by using a linear free energy relation for the rates of quaternary transitions, and by incorporating concepts from recent studies on the physics of geminate rebinding and conformational changes in myoglobin. The model, described by 85 coupled differential equations, quantitatively explains a demanding set of complex kinetic data. Moreover, with the same set of kinetic parameters it simultaneously fits the equilibrium data on ligand binding and the distribution of ligation states. The present results, together with those from single-crystal oxygen binding studies, indicate that the two-state MWC allosteric model has survived its most critical tests.

To determine the speed of communication between protein subunits, time-resolved absorption spectra were measured following partial photodissociation of the carbon monoxide complex of hemoglobin. The experiments were carried out using linearly polarized, 10-ns laser pulses, with the polarization of the excitation pulse both parallel and perpendicular to the polarization of the probe pulse. The substantial contribution to the observed spectra from photoselection effects was eliminated by isotropically averaging the polarized spectra, allowing a detailed comparison of the kinetics as a function of the degree of photolysis. These results show that prior to 1 microsecond both geminate ligand rebinding and conformational relaxation are independent of the number of ligands dissociated from the hemoglobin tetramer, as expected for a two-state allosteric model. After this time the kinetics depend on the ligation state of the tetramer. The conformational relaxation at 10 microseconds can be interpreted in terms of the two-state allosteric model as arising from the R to T quaternary conformational change of both unliganded and singly liganded molecules. These results suggest that communication between subunits requires about 1 microsecond and that the mechanism of the communication which occurs after this time is via the R to T conformational change. The optical anisotropy provides a novel means of accurately determining the extinction coefficients of the transient photoproduct. The decay in the optical anisotropy, moreover, provides an accurate determination of the rotational correlation time of 36 +/- 3 ns.

UV resonance Raman excitation within the S2, S3, and S4 π→π* electronic transitions of pyrene results in strong enhancement of totally symmetric ring vibrations. The Raman modes most strongly enhanced within these electronic transitions derive from vibrations exhibiting Franck–Condon vibronic structure in the absorption spectrum. The 1597 cm−1 (b3g) mode shows selective enhancement between the S3 and S4 transitions, and between the S2 and S3 transitions due to Herzberg–Teller coupling between these symmetry-allowed states. The experimentally observed S4 resonance Raman excitation profiles of the totally symmetric pyrene fundamentals agree closely with those calculated using resonance Raman transform theory if non-Condon contributions are included. We see an increased non-Condon contribution with an increasing vibrational frequency, indicating nonadiabatic vibronic interactions. The high incident energy fluxes of the Nd-YAG laser-based excitation source cause saturation of the pyrene Raman intensities since molecular absorption depletes the ground state population. The long-lived S1 singlet excited state bottlenecks relaxation back to the ground electronic state. Formation of pyrene phototransients is also observed with high incident energy fluxes at particular excitation wavelengths.

Polarized photolysis experiments have been performed on the carbon monoxide complex of myoglobin to assess the effects of photoselection on the kinetics of ligand rebinding and to investigate the reorientational dynamics of the heme plane. The results are analyzed in terms of the optical theory developed in the preceding paper by Ansari and Szabo. Changes in optical density arising from rotational diffusion of the photoselected population produce large deviations from the true geminate ligand rebinding curves if measurements are made with only a single polarization. The apparent ligand rebinding curves are significantly distorted even at photolysis levels greater than 90%. These deviations are eliminated by obtaining isotropically-averaged optical densities from measurements using both parallel and perpendicular polarizations of the probe pulse. These experiments also yield the optical anisotropy, which gives a novel method for accurately determining the degree of photolysis, as well as important information on the reorientational dynamics of the heme plane. The correlation time for the overall rotational diffusion of the molecule is obtained from the decay of the anisotropy. The anisotropy prior to rotational diffusion is lower than that predicted for a rigidly attached, perfectly circular absorber, corresponding to an apparent order parameter of S = 0.95 +/- 0.02. Polarized absorption data on single crystals suggest that the decreased anisotropy results more from internal motions of the heme plane which take place on time scales shorter than the duration of the laser pulse (10 ns) than from out-of-plane polarized transitions.

The kinetics of conformational changes in trout hemoglobin I have been characterized over the temperature range 2-65 degrees C from time-resolved absorption spectra measured following photodissociation of the carbon monoxide complex. Changes in the spectra of the deoxyheme photoproduct were used to monitor changes in the protein conformation. Although the deoxyheme spectral changes are only about 8% of the total spectral change due to ligand rebinding, a combination of high-precision measurements and singular value decomposition of the data permits a detailed analysis of both their amplitudes and relaxation rates. Systematic variation of the degree of photolysis was used to alter the distribution of liganded tetramers, permitting the assignment of the spectral relaxation at 20 microseconds to the R----T quaternary conformational change of the zero-liganded and singly liganded molecules and spectral relaxations at about 50 ns and 2 microseconds to tertiary conformational changes within the R structure. Analysis of the effect of photoselection by the linearly polarized excitation pulse indicates that a major contribution to the apparent geminate rebinding in the 50-ns relaxation arises from rotational diffusion of molecules containing unphotolyzed heme-CO complexes. The activation enthalpy and activation entropy for the R0----T0 transition are +7.4 kcal/mol and -12 cal mol-1 K-1. Using the equilibrium data, delta H = +29.4 kcal/mol and delta S = +84.4 cal mol-1 K-1 [Barisas, B. G., & Gill, S. J. (1979) Biophys. Chem. 9, 235-244], the activation parameters for the T0----R0 transition are calculated to be delta H = +37 kcal/mol and delta S = +73 cal mol-1 K-1. The similarity of the equilibrium and activation parameters for the T0----R0 transition indicates that the transition state is much more R-like than T-like. This result suggests that in the path from T0 to R0 the subunits have already almost completely rearranged into the R configuration when the transition state is reached, while in the path from R0 to T0 the subunits remain in a configuration close to R in the transition state. The finding of an R-like transition state explains why the binding of ligands causes much smaller changes in the R----T rates than in the T----R rates.