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W A Eaton

Publications and source records attributed to W A Eaton.

At least 19 recordsLinked to original sources
PubMed1995

Protein reaction kinetics in a room-temperature glass.

Protein reaction kinetics in aqueous solution at room temperature are often simplified by the thermal averaging of conformational substates. These substates exhibit widely varying reaction rates that are usually exposed by trapping in a glass at low temperature. Here, it is shown that the solvent viscosity, rather than the low temperature, is primarily responsible for the trapping. This was demonstrated by placement of myoglobin in a glass at room temperature and subsequent observation of inhomogeneous reaction kinetics. The high solvent viscosity slowed the rate of crossing the energy barriers that separated the substates and also suppressed any change in the average protein conformation after ligand dissociation.

Binding Sites↗
PubMed1995

Comments on the physics and chemistry of trehalose as a storage medium for hemoglobin-based blood substitutes: "from Kramers Theory to the Battlefield".

A glass of the naturally-occurring sugar trehalose may be a suitable medium for the storage of hemoglobin-based blood substitutes. Trehalose has many or possibly all of the properties required for this purpose, including solubilization of hemoglobin to a very high concentration, lack of toxicity, slowing of oxidation to the non-oxygen binding methemoglobin, stability at room temperature and above, and ease of transport. It should also be possible to prepare hemoglobin extremely rapidly for injection into the circulation in situations where blood replacement is required immediately, as in a domestic emergency room or on the battlefield. These practical considerations are briefly discussed, as well as the theoretical reasons for slowing of chemical reactions in the glassy state.

Blood Substitutes↗
PubMed1994

Conformational relaxation and ligand binding in myoglobin.

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.

Animals↗
PubMed1993

Fast events in protein folding initiated by nanosecond laser photolysis.

Initiation of protein folding by light can dramatically improve the time resolution of kinetic studies. Here we present an example of an 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 closer 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.

Amino Acid Sequence↗
PubMed1993

Simulation of the kinetics of ligand binding to a protein by molecular dynamics: geminate rebinding of nitric oxide to myoglobin.

We have begun to use molecular dynamics to simulate the kinetics of nitric oxide rebinding to myoglobin after photodissociation. Rebinding was simulated using a potential function that switches smoothly between a nonbinding potential and a binding potential as a function of the position and orientation of the ligand, with no barrier arising from the crossing of potential surfaces of different electron spin. In 96 of 100 trajectories, the ligand rebound in < 15 ps. The kinetic progress curve was obtained by determining the time in each trajectory at which the ligand rebound and then calculating the fraction of unbound ligands as a function of time. The curve can be well reproduced by a simple model based on the dynamics of a Langevin particle moving on a one-dimensional potential of mean force calculated from nonreactive protein trajectories. The rate of escape from the energy well adjacent to the heme is in good agreement with the value calculated from experimental data, suggesting that a multiple-well model provides a plausible explanation for the nonexponential rebinding kinetics. A transition-state analysis suggests that protein conformational relaxation coupled to the displacement of the iron from the heme plane is an unlikely cause for the nonexponential rebinding of nitric oxide.

Computer Simulation↗
PubMed1993

Oxygen binding by single crystals of hemoglobin.

Reversible oxygen binding curves for single crystals of hemoglobin in the T quaternary structure have been measured using microspectrophotometry. Saturations were determined from complete visible spectra measured with light linearly polarized parallel to the a and c crystal axes. Striking differences were observed between the binding properties of hemoglobin in the crystal and those of hemoglobin in solution. Oxygen binding to the crystal is effectively noncooperative, the Bohr effect is absent, and there is no effect of chloride ion. Also, the oxygen affinity is lower than that of the T quaternary structure in solution. The absence of the Bohr effect supports Perutz's hypothesis on the key role of the salt bridges, which are known from X-ray crystallography to remain intact upon oxygenation. The low affinity and absence of the Bohr effect can be explained by a generalization of the MWC-PSK model (Monod, Wyman, & Changeux, 1965; Perutz, 1970; Szabo & Karplus, 1972) in which both high- and low-affinity tertiary conformations, with broken and unbroken salt bridges, respectively, are populated in the T quaternary structure. Because the alpha and beta hemes make different projections onto the two crystal axes, separate binding curves for the alpha and beta subunits could be calculated from the two measured binding curves. The approximately 5-fold difference between the oxygen affinities of the alpha and beta subunits is much smaller than that predicted from the crystallographic study of Dodson, Liddington, and co-workers, which suggested that oxygen binds only to the alpha hemes.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystallization↗
PubMed1993

Photoselection in polarized photolysis experiments on heme proteins.

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.

Animals↗
PubMed1992

Speed of intersubunit communication in proteins.

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.

Carboxyhemoglobin↗
PubMed1992

The role of solvent viscosity in the dynamics of protein conformational changes.

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."

Carbon Monoxide↗
PubMed1991

Dynamics of the quaternary conformational change in trout hemoglobin.

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.

Amino Acid Sequence↗
PubMed1991

Crystals of haemoglobin with the T quaternary structure bind oxygen noncooperatively with no Bohr effect.

The relationship between the structure and function of haemoglobin has mainly been studied by comparing its X-ray crystal structures with its function in solutions. To make a direct comparison we have studied the functional properties of haemoglobin in single crystals, an approach that has been an important part of the investigation of several enzyme mechanisms. Here we report on the oxygen binding by single crystals of human haemoglobin grown in solutions of polyethylene glycol. Unlike haemoglobin crystals formed in concentrated salt solution, which crack and become disordered on oxygenation, crystals grown in polyethylene glycol remain intact. X-ray studies have shown that the T (deoxy) quaternary structure of haemoglobin in this crystal at pH 7.0 is maintained at atmospheric oxygen pressure, and that the salt-bridges are not broken. We find striking differences between oxygen binding by haemoglobin in this crystal and by haemoglobin in solution. Not only is oxygenation of the crystal noncooperative, but the oxygen affinity is independent of pH in the range 6.0-8.5, and is much lower than that of the T state in solution. The lack of cooperativity without a change in quaternary structure is predicted by the two-state allosteric model of Monod, Wyman and Changeux. The absence of a Bohr effect without breakage of salt-bridges is predicted by Perutz's stereochemical mechanism. In contrast to the X-ray result that oxygen binds only to the alpha haems, our measurements show that the alpha haems have only a slightly higher affinity than the beta haems.

Allosteric Regulation↗
PubMed1991

Application of linear free energy relations to protein conformational changes: the quaternary structural change of hemoglobin.

The transition state for the R in equilibrium with T quaternary conformational change of hemoglobin has thermodynamic properties much closer to those of the R conformation than to those of the T conformation. This finding is based on a comparison of activation and equilibrium enthalpy and entropy changes and on the observation of a linear free energy relationship between quaternary rate and equilibrium constants. A previous theoretical study [Janin, J. & Wodak, S. J. (1985) Biopolymers 24, 509-526], using a highly simplified energy function, suggests that the R-like transition state is the result of a reaction pathway with the maximum buried surface area between alpha beta dimers.

Allosteric Regulation↗
PubMed1988

Time-resolved optical spectroscopy and structural dynamics following photodissociation of carbonmonoxyhemoglobin.

A summary is presented of our current understanding of the kinetics of ligand rebinding and conformational changes at room temperature following photodissociation of the carbon monoxide complex of hemoglobin with pulsed lasers. The events which occur subsequent to excitation have been followed over 12 decades in time, from about 100 fs to the completion of ligand rebinding at about 100 ms. Experiments with picosecond and subpicosecond lasers by others, together with molecular dynamics simulations, indicate that by 1 ns the deoxyhemoglobin photoproduct is in a thermally equilibrated ground electronic state, so that subsequent processes are unaffected by the initial laser excitation. The principal results have been obtained from time-resolved optical absorption spectroscopy using a sensitive nanosecond laser spectrometer. Five relaxations have been observed which are interpreted as geminate rebinding at about 50 ns that competes with motion of the ligand away from the heme which produces a tertiary conformational change, a second tertiary conformational change at 0.5-1 microseconds, transition from the R to T quaternary structure at about 20 microseconds, and overall bimolecular rebinding of ligands from the solvent to the R and T quaternary structures at about 200 microseconds and 10 ms. Assuming that the dissociation pathway in photolysis experiments is the reverse of the association pathway, we find that for the R state there is a 40% probability that the ligand will bind to the heme after entering the protein, and a 60% probability that it will return to the solvent. Studies on the alpha-subunit of an iron-cobalt hybrid hemoglobin indicate that carbon monoxide enters the protein at the same rate for both R and T quaternary structures. For the alpha-subunit in the T state the probability of binding after entry is much lower, and the ligand returns to the solvent more than 99% of the time, accounting for the 60-fold overall lower association rate. This decreased probability of binding results from a decreased rate of binding to the heme from within the protein, and not an increased rate of return to the solvent. There are still unresolved problems on the basic structural description of carbon monoxide binding and dissociation, particularly the functional significance of the tertiary relations in both the R and T states, and the precise number of kinetic barriers within the protein.

Algorithms↗
PubMed1988

Picosecond transient absorption study of photodissociated carboxy hemoglobin and myoglobin.

The optical transient absorption spectra at 30 ps and 6.5 ns after photolysis are compared for both carboxy hemoglobin (HbCO) and carboxy myoglobin (MbCO). Both 355- and 532-nm excitation pulses were used. In all cases the shapes of the optical difference spectra thus generated are stationary over the complete time-scale studied. The photolysis spectra for MbCO are not significantly different from the equilibrium difference spectra generated on the same picosecond spectrometer when measured to an accuracy of +/- 0.5 nm. In addition, spectral parameters for delegated HbCO generated on the same spectrometer but detected by two different techniques, either by a Vidicon detector or point by point with photomultiplier tubes, are reported; the results are different from some of the previously reported picosecond experiments.

Animals↗
PubMed1988

The effect of quaternary structure on the kinetics of conformational changes and nanosecond geminate rebinding of carbon monoxide to hemoglobin.

To determine the effect of quaternary structure on the individual kinetic steps in the binding of carbon monoxide to the alpha subunit of hemoglobin, time-resolved absorption spectra were measured after photodissociation of carbon monoxide from a hemoglobin tetramer in which cobalt was substituted for iron in the beta subunits. Cobalt porphyrins do not bind carbon monoxide. Spectra were measured in the Soret region at room temperature after time delays that varied from a few nanoseconds to the completion of ligand rebinding at about 100 ms. The results show that the liganded molecule, alpha(Fe-CO)2 beta(Co)2, is in the R state, but can be almost completely switched into the T state by the allosteric effectors inositol hexaphosphate and bezafibrate. The geminate yield, which is the probability that the ligand rebinds to the heme from within the protein, is found to be 40% for the R state and less than 1% for the T state. According to the simplest kinetic model, these results indicate that carbon monoxide enters the protein in the R and T quaternary conformations at the same rate, and that the 60-fold decrease in the overall binding rate, of carbon monoxide to the alpha subunit in the T state compared to the R state is almost completely accounted for by the decreased probability of binding after the ligand has entered the protein. The results further suggest that the low probability for the T state results from a decreased binding rate to the heme and not from an increased rate of return of the ligand to the solvent.

Allosteric Regulation↗