Biomedical subjects

William A Eaton

Publications and source records attributed to William A Eaton.

16 recordsLinked to original sources

PubMed2006

Folding and aggregation kinetics of a beta-hairpin.

We have investigated the solution structure, equilibrium properties, and folding kinetics of a 17-residue beta-hairpin-forming peptide derived from the protein ubiquitin. NMR experiments show that at 4 degrees C the peptide has a highly populated beta-hairpin conformation. At protein concentrations higher than 0.35 mM, the peptide aggregates. Sedimentation equilibrium measurements show that the aggregate is a trimer, while NMR indicates that the beta-hairpin conformation is maintained in the trimer. The relaxation kinetics in nanosecond laser temperature-jump experiments reveal a concentration-independent microsecond phase, corresponding to beta-hairpin unfolding-refolding, and a concentration-dependent millisecond phase due to oligomerization. Kinetic modeling of the relaxation rates and amplitudes yields the folding and unfolding rates for the monomeric beta-hairpin, as well as assembly and disassembly rates for trimer formation consistent with the equilibrium constant determined by sedimentation equilibrium. When the net charge on the peptides and ionic strength were taken into account, the rate of trimer assembly approaches the Debye-Smoluchowski diffusion limit. At 300 K, the rate of formation of the monomeric hairpin is (17 micros)(-1), compared to rates of (0.8 micros)(-1) to (52 micros)(-1) found for other peptides. After using Kramers theory to correct for the temperature dependence of the pre-exponential factor, the activation energy for hairpin formation is near zero, indicating that the barrier to folding is purely entropic. Comparisons with previously measured rates for a series of hairpins are made to distinguish between zipper and hydrophobic collapse mechanisms. Overall, the experimental data are most consistent with the zipper mechanism in which structure formation is initiated at the turn, the mechanism predicted by the Ising-like statistical mechanical model that was developed to explain the equilibrium and kinetic data for the beta-hairpin from protein GB1. In contrast, the majority of simulation studies favor a hydrophobic collapse mechanism. However, with few exceptions, there is little or no quantitative comparison of the simulation results with experimental data.

Amino Acid Sequence↗

PubMed2006

Effects of denaturants on the dynamics of loop formation in polypeptides.

Quenching of the triplet state of tryptophan by close contact with cysteine has been used to measure the reaction-limited and diffusion-limited rates of loop formation in disordered polypeptides having the sequence cys-(ala-gly-gln)j-trp (j=1-9). The decrease in the length-dependence of the reaction-limited rate for short chains in aqueous buffer, previously attributed to chain stiffness, is not observed at high concentrations of chemical denaturant (6 M GdmCl and 8 M urea), showing that denaturants increase chain flexibility. For long chains, both reaction-limited and diffusion-limited rates are significantly smaller in denaturant and exhibit a steeper length dependence. The results can be explained using end-to-end distributions from a wormlike chain model in which excluded volume interactions are incorporated by associating a 0.4-0.5 nm diameter hard sphere with the end of each virtual peptide bond. Fitting the data with this model shows that the denaturants reduce the persistence length from approximately 0.6 nm to approximately 0.4 nm, only slightly greater than the length of a peptide bond. The same model also describes the reported length dependence for the radii of gyration of chemically denatured proteins containing 50-400 residues. The end-to-end diffusion coefficients obtained from the diffusion-limited rates are smaller than the sum of the monomer diffusion coefficients and exhibit significant temperature dependence, suggesting that diffusion is slowed by internal friction arising from barriers to backbone conformational changes.

Computer Simulation↗

PubMed2006

Sub-microsecond protein folding.

We have investigated the structure, equilibria, and folding kinetics of an engineered 35-residue subdomain of the chicken villin headpiece, an ultrafast-folding protein. Substitution of two buried lysine residues by norleucine residues stabilizes the protein by 1 kcal/mol and increases the folding rate sixfold, as measured by nanosecond laser T-jump. The folding rate at 300 K is (0.7 micros)(-1) with little or no temperature dependence, making this protein the first sub-microsecond folder, with a rate only twofold slower than the theoretically predicted speed limit. Using the 70 ns process to obtain the effective diffusion coefficient, the free energy barrier height is estimated from Kramers theory to be less than approximately 1 kcal/mol. X-ray crystallographic determination at 1A resolution shows no significant change in structure compared to the single-norleucine-substituted molecule and suggests that the increased stability is electrostatic in origin. The ultrafast folding rate, very accurate X-ray structure, and small size make this engineered villin subdomain an ideal system for simulation by atomistic molecular dynamics with explicit solvent.

Amino Acid Substitution↗

PubMed2005

High-resolution x-ray crystal structures of the villin headpiece subdomain, an ultrafast folding protein.

The 35-residue subdomain of the villin headpiece (HP35) is a small ultrafast folding protein that is being intensely studied by experiments, theory, and simulations. We have solved the x-ray structures of HP35 and its fastest folding mutant [K24 norleucine (nL)] to atomic resolution and compared their experimentally measured folding kinetics by using laser temperature jump. The structures, which are in different space groups, are almost identical to each other but differ significantly from previously solved NMR structures. Hence, the differences between the x-ray and NMR structures are probably not caused by lattice contacts or crystal/solution differences, but reflect the higher accuracy of the x-ray structures. The x-ray structures reveal important details of packing of the hydrophobic core and some additional features, such as cross-helical H bonds. Comparison of the x-ray structures indicates that the nL substitution produces only local perturbations. Consequently, the finding that the small stabilization by the mutation is completely reflected in an increased folding rate suggests that this region of the protein is as structured in the transition state as in the folded structure. It is therefore a target for engineering to increase the folding rate of the subdomain from approximately 0.5 micros(-1) for the nL mutant to the estimated theoretical speed limit of approximately 3 micros(-1).

Crystallography, X-Ray↗

PubMed2005

Determination of ultrafast protein folding rates from loop formation dynamics.

Quenching of the triplet state of tryptophan by contact with cysteine can be used to measure the kinetics of loop formation in unfolded proteins. Here we show that cysteine quenching dynamics also provide a novel method for measuring folding rates when the exchange between folded and unfolded states is faster than the unquenched triplet lifetime (approximately 100 micros). We use this technique to investigate folding/unfolding kinetics of the 35 residue headpiece subdomain of the protein villin, which contains a single tryptophan residue and was engineered to contain a cysteine residue at the N terminus. At intermediate concentrations of denaturant the time-course of the triplet decay consists of two relaxations, the rates and amplitudes of which reveal the fast kinetics for folding and unfolding of this protein. The folding rates extracted using a simple kinetic model are close to those reported previously from laser-induced temperature-jump experiments that employ the change in tryptophan fluorescence as a probe. However, the results differ significantly from those reported from dynamic NMR line shape analysis on a variant with methionine at the N terminus, an issue that remains to be resolved. The analysis of the triplet quenching kinetics also shows that the quenching rates in the unfolded state increase with decreasing denaturant concentration, indicating a compaction of the unfolded protein.

Antioxidants↗

PubMed2005

Polyproline and the "spectroscopic ruler" revisited with single-molecule fluorescence.

To determine whether Forster resonance energy transfer (FRET) measurements can provide quantitative distance information in single-molecule fluorescence experiments on polypeptides, we measured FRET efficiency distributions for donor and acceptor dyes attached to the ends of freely diffusing polyproline molecules of various lengths. The observed mean FRET efficiencies agree with those determined from ensemble lifetime measurements but differ considerably from the values expected from Forster theory, with polyproline treated as a rigid rod. At donor-acceptor distances much less than the Forster radius R(0), the observed efficiencies are lower than predicted, whereas at distances comparable to and greater than R(0), they are much higher. Two possible contributions to the former are incomplete orientational averaging during the donor lifetime and, because of the large size of the dyes, breakdown of the point-dipole approximation assumed in Forster theory. End-to-end distance distributions and correlation times obtained from Langevin molecular dynamics simulations suggest that the differences for the longer polyproline peptides can be explained by chain bending, which considerably shortens the donor-acceptor distances.

Fluorescence↗

PubMed2004

Understanding the shape of sickled red cells.

To understand the physical basis of the wide variety of shapes of deoxygenated red cells from patients with sickle cell anemia, we have measured the formation rate and volume distribution of the birefringent domains of hemoglobin S fibers. We find that the domain formation rate depends on the approximately 80th power of the protein concentration, compared to approximately 40th power for the concentration dependence of the reciprocal of the delay time that precedes fiber formation. These remarkably high concentration dependences, as well as the exponential distribution of domain volumes, can be explained by the previously proposed double nucleation model in which homogeneous nucleation of a single fiber triggers the formation of an entire domain via heterogeneous nucleation and growth. The enormous sensitivity of the domain formation rate to intracellular hemoglobin S concentration explains the variable cell morphology and why rapid polymerization results in cells that do not appear sickled at all.

Anemia, Sickle Cell↗

PubMed2004

New insights into allosteric mechanisms from trapping unstable protein conformations in silica gels.

To understand why the classical two-state allosteric model of Monod, Wyman, and Changeux explains cooperative oxygen binding by hemoglobin but does not explain changes in oxygen affinity by allosteric inhibitors, we have investigated the kinetic properties of unstable conformations transiently trapped by encapsulation in silica gels. Conformational trapping reveals that after nanosecond photodissociation of carbon monoxide a large fraction of the subunits of the T quaternary structure has kinetic properties almost identical to those of subunits of the R quaternary structure. Addition of allosteric inhibitors reduces both the fraction of R-like subunits and the oxygen affinity of the T quaternary structure. These kinetic and equilibrium results are readily explained by a recently proposed generalization of the Monod-Wyman-Changeux model in which a pre-equilibrium between two functionally different tertiary, rather than quaternary, conformations plays the central role.

Allosteric Regulation↗

PubMed2004

The protein folding 'speed limit'.

How fast can a protein possibly fold? This question has stimulated experimentalists to seek fast folding proteins and to engineer them to fold even faster. Proteins folding at or near the speed limit are prime candidates for all-atom molecular dynamics simulations. They may also have no free energy barrier, allowing the direct observation of intermediate structures on the pathways from the unfolded to the folded state. Both experimental and theoretical approaches predict a speed limit of approximately N/100micros for a generic N-residue single-domain protein, with alpha proteins folding faster than beta or alphabeta. The predicted limits suggest that most known ultrafast folding proteins can be engineered to fold more than ten times faster.

Computer Simulation↗

PubMed2003

Kinetics of intramolecular contact formation in a denatured protein.

Quenching of the triplet state of tryptophan by cysteine has provided a new tool for measuring the rate of forming a specific intramolecular contact in disordered polypeptides. Here, we use this technique to investigate contact formation in the denatured state of CspTm, a small cold-shock protein from Thermotoga maritima, engineered to contain a single tryptophan residue (W29) and a single cysteine residue at the C terminus (C67). At all concentrations of denaturant, the decay rate of the W29 triplet of the unfolded protein is more than tenfold faster than the rate observed for the native protein ( approximately 10(4)s(-1)). Experiments on the unfolded protein without the added C-terminal cysteine residue show that this faster rate results entirely from contact quenching by C67. The quenching rate in the unfolded state by C67 increases at concentrations of denaturant that favor folding, indicating a compaction of the unfolded protein as observed previously in single-molecule Förster resonance energy transfer (FRET) experiments.

Bacterial Proteins↗

PubMed2003

Single-molecule measurement of protein folding kinetics.

In order to investigate the behavior of single molecules under conditions far from equilibrium, we have coupled a microfabricated laminar-flow mixer to a confocal optical system. This combination enables time-resolved measurement of Förster resonance energy transfer after an abrupt change in solution conditions. Observations of a small protein show the evolution of the intramolecular distance distribution as folding progresses. This technique can expose subpopulations, such as unfolded protein under conditions favoring the native structure, that would be obscured in equilibrium experiments.

Bacterial Proteins↗

PubMed2003

Experimental tests of villin subdomain folding simulations.

We have used laser temperature-jump to investigate the kinetics and mechanism of folding the 35 residue subdomain of the villin headpiece. The relaxation kinetics are biphasic with a sub-microsecond phase corresponding to a helix-coil transition and a slower microsecond phase corresponding to overall unfolding/refolding. At 300 K, the folding time is 4.3(+/-0.6) micros, making it the fastest folding, naturally occurring protein, with a rate close to the theoretical speed limit. This time is in remarkable agreement with the prediction of 5 (+11,-3) micros by Zagrovic et al. from atomistic molecular dynamics simulations using an implicit solvent model. We test their prediction that replacement of the C-terminal phenylalanine residue with alanine will increase the folding rate by removing a transient non-native interaction. We find that the alanine substitution has no effect on the folding rate or on the equilibrium constant. Implications of this result for the validity of the simulated folding mechanism are discussed.

Alanine↗

PubMed2003

Linus Pauling and sickle cell disease.

The 1949 paper by Linus Pauling et al. [Science 110 (1949) 543-548] describing the discovery of sickle cell anemia as the first molecular disease had a major impact on biology and medicine. Inspired by the scholarly works of John Edsall on the history of hemoglobin research, I present a brief retrospective analysis of Pauling's paper. This is followed by some personal recollections of Edsall and Pauling.

Anemia, Sickle Cell↗

PubMed2002

Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy.

Protein folding is inherently a heterogeneous process because of the very large number of microscopic pathways that connect the myriad unfolded conformations to the unique conformation of the native structure. In a first step towards the long-range goal of describing the distribution of pathways experimentally, Förster resonance energy transfer (FRET) has been measured on single, freely diffusing molecules. Here we use this method to determine properties of the free-energy surface for folding that have not been obtained from ensemble experiments. We show that single-molecule FRET measurements of a small cold-shock protein expose equilibrium collapse of the unfolded polypeptide and allow us to calculate limits on the polypeptide reconfiguration time. From these results, limits on the height of the free-energy barrier to folding are obtained that are consistent with a simple statistical mechanical model, but not with the barriers derived from simulations using molecular dynamics. Unlike the activation energy, the free-energy barrier includes the activation entropy and thus has been elusive to experimental determination for any kinetic process in solution.

Bacterial Proteins↗

PubMed2002

A tertiary two-state allosteric model for hemoglobin.

The two-state allosteric model of Monod, Wyman, and Changeux (MWC) provides an excellent description of homotropic effects in a vast array of equilibrium and kinetic measurements on cooperative ligand binding by hemoglobin. However, in contrast to experimental observations, the model does not allow for alteration of the ligand affinity of the T quaternary structure by allosteric effectors. This failure to explain heterotropic effects has been appreciated for over 30 years, and it has been generally assumed to result from tertiary conformational changes in the absence of a quaternary change. Here we explore a model that preserves the essential MWC idea that binding without a quaternary conformational change is non-cooperative, but where tertiary conformations of individual subunits play the primary role instead of the quaternary conformations. In this model, which we call the 'tertiary two-state (TTS) model', the two affinity states correspond to two tertiary conformations of individual subunits rather than the two quaternary conformations of the MWC two-state allosteric model. Ligation and the R quaternary structure bias the subunit population toward the high affinity tertiary conformation, while deligation and the T quaternary structure favor the low affinity tertiary conformation. We show that the model is successful in quantitatively explaining a demanding set of kinetic data from nanosecond carbon monoxide photodissociation experiments at times longer than approximately 1 micros. Better agreement between the model and the submicrosecond kinetic data may result from detailed considerations of the distribution and dynamics of conformational substates of the two tertiary conformations. The model is consistent with the results of solution, gel, and single crystal oxygen binding studies, but underestimates the population of doubly-liganded molecules determined in low-temperature electrophoresis experiments.

Allosteric Site↗

PubMed2002

Measuring dynamic flexibility of the coil state of a helix-forming peptide.

To investigate the dynamic flexibility of the coil state of a helix-forming peptide the end-to-end contact rate was determined. Nanosecond optical excitation of tryptophan at one end of a 22 residue, alanine peptide populates a long-lived triplet state which is quenched upon close contact with a cyclic disulfide attached to the opposite end. Analysis of the decay of the triplet population using a two-state model for helix formation yields the diffusion-limited end-to-end contact rate of the coil state of the peptide as well as the helix-->coil and coil-->helix rates. The helix-coil rates are very similar to those previously measured in laser temperature-jump experiments. The end-to-end contact rate of 1.1 x 10(7) s(-1) in the coil state is tenfold faster than the rate for a disordered peptide with threonine substituted for alanine and, somewhat surprisingly, is about twice the rate for a disordered glycine-containing peptide. These differences are discussed in terms of the theory of Szabo, Schulten and Schulten. The rates should provide important new benchmarks for testing the accuracy of atomistic molecular dynamics simulations.

Disulfides↗