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J Hofrichter

Publications and source records attributed to J Hofrichter.

At least 19 recordsLinked to original sources
PubMed1998

A statistical mechanical model for beta-hairpin kinetics.

Understanding the mechanism of protein secondary structure formation is an essential part of the protein-folding puzzle. Here, we describe a simple statistical mechanical model for the formation of a beta-hairpin, the minimal structural element of the antiparallel beta-pleated sheet. The model accurately describes the thermodynamic and kinetic behavior of a 16-residue, beta-hairpin-forming peptide, successfully explaining its two-state behavior and apparent negative activation energy for folding. The model classifies structures according to their backbone conformation, defined by 15 pairs of dihedral angles, and is further simplified by considering only the 120 structures with contiguous stretches of native pairs of backbone dihedral angles. This single sequence approximation is tested by comparison with a more complete model that includes the 2(15) possible conformations and 15 x 2(15) possible kinetic transitions. Finally, we use the model to predict the equilibrium unfolding curves and kinetics for several variants of the beta-hairpin peptide.

Animals↗
PubMed1997

Folding dynamics and mechanism of beta-hairpin formation.

Protein chains coil into alpha-helices and beta-sheet structures. Knowing the timescales and mechanism of formation of these basic structural elements is essential for understanding how proteins fold. For the past 40 years, alpha-helix formation has been extensively investigated in synthetic and natural peptides, including by nanosecond kinetic studies. In contrast, the mechanism of formation of beta structures has not been studied experimentally. The minimal beta-structure element is the beta-hairpin, which is also the basic component of antiparallel beta-sheets. Here we use a nanosecond laser temperature-jump apparatus to study the kinetics of folding a beta-hairpin consisting of 16 amino-acid residues. Folding of the hairpin occurs in 6 micros at room temperature, which is about 30 times slower than the rate of alpha-helix formation. We have developed a simple statistical mechanical model that provides a structural explanation for this result. Our analysis also shows that folding of a beta-hairpin captures much of the basic physics of protein folding, including stabilization by hydrogen bonding and hydrophobic interactions, two-state behaviour, and a funnel-like, partially rugged energy landscape.

Kinetics↗
PubMed1997

Laser temperature jump study of the helix<==>coil kinetics of an alanine peptide interpreted with a 'kinetic zipper' model.

The kinetics of the helix<==>coil transition of an alanine-based peptide following a laser-induced temperature jump were monitored by the fluorescence of an N-terminal probe, 4-(methylamino)benzoic acid (MABA). This probe forms a peptide hydrogen bond to the helix backbone, which changes its fluorescence quantum yield. The MABA fluorescence intensity decreases in a single exponential relaxation, with relaxation times that are weakly temperature dependent, exhibiting a maximum value of approximately 20 ns near the midpoint of the melting transition. We have developed a new model, the kinetic version of the equilibrium 'zipper' model for helix<==>coil transitions to explain these results. In this 'kinetic zipper' model, an enormous reduction in the number of possible species results from the assumption that each molecule contains either no helical residues or a single contiguous region of helix (the single-sequence approximation). The decay of the fraction of N-terminal residues that are helical, calculated from numerical solutions of the kinetic equations which describe the model, can be approximately described by two exponential relaxations having comparable amplitudes. The shorter relaxation time results from rapid unzipping (and zipping) of the helix ends in response to the temperature jump, while the longer relaxation time results from equilibration of helix-containing and non-helix-containing structures by passage over the nucleation free energy barrier. The decay of the average helix content is dominated by the slower process. The model therefore explains the experimental observation that relaxation for the N-terminal fluorescent probe is approximately 8-fold faster than that for the infrared probe of Williams et al. [(1996) Biochemistry 35, 691-697], which measures the average helix content, but does not account for the absence of observable amplitude for the slow relaxation in the fluorescence experiments (<10% slow phase). If we assume that the activation barrier for the coil-->helix rate is purely entropic, the model can also explain the maximum in the temperature dependence of the relaxation time for the fluorescent probe. Parameters that best reproduce the melting curves and the ratio of relaxation times predict a value of the cooperativity parameter sigma which is approximately 3-fold larger than previously reported values obtained from fitting equilibrium data only. The helix growth rate of approximately 10(8) s-1 that reproduces the experimental relaxation times is approximately 100-fold slower than those observed in molecular dynamics simulations. These parameters can be used to simulate the kinetically cooperative formation of a helix from the all-coil state.

Alanine↗
PubMed1997

Can a two-state MWC allosteric model explain hemoglobin kinetics?

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.

Allosteric Site↗
PubMed1997

Submillisecond protein folding kinetics studied by ultrarapid mixing.

An ultrarapid-mixing continuous-flow method has been developed to study submillisecond folding of chemically denatured proteins. Turbulent flow created by pumping solutions through a small gap dilutes the denaturant in tens of microseconds. We have used this method to study cytochrome c folding kinetics in the previously inaccessible time range 80 micros to 3 ms. To eliminate the heme-ligand exchange chemistry that complicates and slows the folding kinetics by trapping misfolded structures, measurements were made with the imidazole complex. Fluorescence quenching due to excitation energy transfer from the tryptophan to the heme was used to monitor the distance between these groups. The fluorescence decrease is biphasic. There is an unresolved process with tau < 50 micros, followed by a slower, exponential process with tau = 600 micros at the lowest denaturant concentration (0.2 M guanidine hydrochloride). These kinetics are interpreted as a barrier-free, partial collapse to the new equilibrium unfolded state at the lower denaturant concentration, followed by slower crossing of a free energy barrier separating the unfolded and folded states. The results raise several fundamental issues concerning the dynamics of collapse and barrier crossings in protein folding.

Animals↗
PubMed1997

Submillisecond kinetics of protein folding.

New experimental methods permit observation of protein folding and unfolding on the previously inaccessible nanosecond-microsecond timescale. These studies are beginning to establish times for the elementary motions in protein folding - secondary structure and loop formation, local hydrophobic collapse, and global collapse to the compact denatured state. They permit an estimate of about one microsecond for the shortest time in which a protein can possibly fold.

Humans↗
PubMed1996

A multiparameter analysis of sickle erythrocytes in patients undergoing hydroxyurea therapy.

During 24 weeks of hydroxyurea treatment, we monitored red blood cell (RBC) parameters in three patients with sickle cell disease, including F-cell and F-reticulocyte profiles, distributions of delay times for intracellular polymerization, sickle erythrocyte adherence to human umbilical vein endothelial cells in a laminar flow chamber, RBC phthalate density profiles, mean corpuscular hemoglobin concentration and cation content, reticulocyte mean corpuscular hemoglobin concentration, 1H-nuclear magnetic resonance transverse relaxation rates of packed RBCs, and plasma membrane lateral and rotational mobilities of band 3 and glycophorins. Hydroxyurea increases the fraction of cells with sufficiently long delay times to escape the microcirculation before polymerization begins. Furthermore, high pretreatment adherence to human umbilical vein endothelial cells of sickle RBCs decreased to normal after only 2 weeks of hydroxyurea treatment, preceding the increase in fetal hemoglobin levels. The lower adhesion of sickle RBCs to endothelium would facilitate escape from the microcirculation before polymerization begins. Hydroxyurea shifted several biochemical and biophysical parameters of sickle erythrocytes toward values observed with hemoglobin SC disease, suggesting that hydroxyurea moderates sickle cell disease toward the milder, but still clinically significant, hemoglobin SC disease. The 50% reduction in sickle crises documented in the Multicenter Study of Hydroxyurea in Sickle Cell Disease is consistent with this degree of erythrocyte improvement.

Adult↗
PubMed1996

Fast events in protein folding.

Understanding how proteins fold is one of the central problems in biochemistry. A new generation of kinetic experiments has emerged to investigate the mechanisms of protein folding on the previously inaccessible submillisecond time scale. These experiments provide the first glimpse of processes such as secondary structure formation, local hydrophobic collapse, global collapse to compact denatured states, and fast barrier crossings to the native state. Key results are summarized and discussed in terms of the statistical energy landscape theory of protein folding.

Apoproteins↗
PubMed1996

Diffusion-limited contact formation in unfolded cytochrome c: estimating the maximum rate of protein folding.

How fast can a protein fold? The rate of polypeptide collapse to a compact state sets an upper limit to the rate of folding. Collapse may in turn be limited by the rate of intrachain diffusion. To address this question, we have determined the rate at which two regions of an unfolded protein are brought into contact by diffusion. Our nanosecond-resolved spectroscopy shows that under strongly denaturing conditions, regions of unfolded cytochrome separated by approximately 50 residues diffuse together in 35-40 microseconds. This result leads to an estimate of approximately (1 microsecond)-1 as the upper limit for the rate of protein folding.

Cytochrome c Group↗
PubMed1995

A second generation transgenic mouse model expressing both hemoglobin S (HbS) and HbS-Antilles results in increased phenotypic severity.

We report on a second generation of transgenic mice produced by crossing a transgenic mouse line expressing high levels of human alpha and beta S chains (alpha H beta S [beta MDD]) with a line expressing human alpha and beta S-Antilles (beta SAnt). We hypothesized that mice expressing both hemoglobins (Hbs) would have a more severe phenotype because the reduced oxygen affinity and solubility of the beta S-Antilles might enhance the rate and extent of polymer formation. We obtained mice that expressed both beta S and beta S-Antilles. The doubly transgenic mice that are heterozygous for deletion of mouse beta Major (beta MD) occurred with reduced frequency and those that are homozygous for deletion of mouse beta Major (beta MDD) occurred at a much reduced frequency and suffered early mortality. Human alpha was 58% of all alpha globin for all animals, whereas beta S and beta S-Antilles were 34% and 28% of all beta globins for beta MD mice and 42% and 36% for beta MDD mice. Hematocrit, Hb, and mean corpuscular Hb were normal for all transgenic mice, but reticulocyte levels were higher for the doubly transgenic mice versus alpha H beta S [beta MDD] mice older than 30 days (10.0% +/- 1.0% v 4.3% +/- 0.4%; P < .001, mean +/- SE, n = 20 and n = 10, respectively) and control mice (3.9% +/- 0.4%). Reticulocytosis was more severe in mice less than 30 days old ( > 20% for alpha H beta S beta S-Ant[beta MDD] mice). The median mean corpuscular hemoglobin concentration of doubly transgenic mice was higher than that of alpha H beta S[beta MDD] mice with a variable number of very dense cells. Delay times for polymerization of Hb in red blood cells from alpha H beta S beta S-Ant[beta MDD] mice were shorter than those of alpha H beta S[beta MDD] mice, and there were fewer cells with delay times greater than 100 seconds. Urine-concentrating ability in control mice under ambient conditions is 2,846 +/- 294 mOsm and was reduced 30% to 1,958 +/- 240 mOsm, P < 4 x 10(-8) in all mice expressing both transgenes. We conclude that doubly transgenic mice have a more severe phenotype than either of the two parental lines. These mice may be suitable for validating therapeutic intervention in sickle cell disease.

Anemia, Sickle Cell↗
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↗
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

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↗