Biomedical subjects

T B Woolf

Publications and source records attributed to T B Woolf.

At least 19 recordsLinked to original sources

PubMed2001

Predicting properties of intrinsically unstructured proteins.

There is increasing evidence that intrinsically unstructured proteins or protein domains have important biological functions. These types of proteins may be productively analyzed using polymer theory developed to predict global physical properties of polymers. In these theories molecular detail is "coarse grained" out of the models, and replaced with a small number of parameters that characterize the polymer. This reduction in complexity allows extremely large systems to be studied. In the case of simulations, the time scales accessible also increase significantly. Here we discuss the application of polymer theory to unstructured proteins, and consider how to classify proteins within a polymer framework. We then review polymer theory that is relevant to predicting functionally important properties, such as radius of gyration, height of a polymer brush and force required to compress a polymer brush.

Biopolymers↗

PubMed2000

Efficient dynamic importance sampling of rare events in one dimension.

Exploiting stochastic path-integral theory, we obtain by simulation substantial gains in efficiency for the computation of reaction rates in one-dimensional, bistable, overdamped stochastic systems. Using a well-defined measure of efficiency, we compare implementations of "dynamic importance sampling" (DIMS) methods to unbiased simulation. The best DIMS algorithms are shown to increase efficiency by factors of approximately 20 for a 5k(B)T barrier height and 300 for 9k(B)T, compared to unbiased simulation. The gains result from close emulation of natural (unbiased), instantonlike crossing events with artificially decreased waiting times between events that are corrected for in rate calculations. The artificial crossing events are generated using the closed-form solution to the most probable crossing event described by the Onsager-Machlup action. While the best biasing methods require the second derivative of the potential (resulting from the "Jacobian" term in the action, which is discussed at length), algorithms employing solely the first derivative do nearly as well. We discuss the importance of one-dimensional models to larger systems, and suggest extensions to higher-dimensional systems.

Algorithms↗

PubMed2000

Dipole lattice membrane model for protein calculations.

A dipole lattice model for lipid membranes and their interactions with peptides is presented. It uses the Langevin dipole method to calculate electrostatic interactions in the heterogeneous membrane environment. A series of test cases are presented, including spherical charges, dipoles, side chain analogs, and helical peptides. The model consistently produces qualitatively correct results.

Alamethicin↗

PubMed2000

Modulation of glycophorin A transmembrane helix interactions by lipid bilayers: molecular dynamics calculations.

Starting from the glycophorin A dimer structure determined by NMR, we performed simulations of both dimer and monomer forms in explicit lipid bilayers with constant normal pressure, lateral area, and temperature using the CHARMM potential. Analysis of the trajectories in four different lipids reveals how lipid chain length and saturation modulate the structural and energetic properties of transmembrane helices. Helix tilt, helix-helix crossing angle, and helix accessible volume depend on lipid type in a manner consistent with hydrophobic matching concepts: the most relevant lipid property appears to be the bilayer thickness. Although the net helix-helix interaction enthalpy is strongly attractive, analysis of residue-residue interactions reveals significant unfavorable electrostatic repulsion between interfacial glycine residues previously shown to be critical for dimerization. Peptide volume is nearly conserved upon dimerization in all lipid types, indicating that the monomeric helices pack equally well with lipid as dimer helices do with one another. Enthalpy calculations indicate that the helix-environment interaction energy is lower in the dimer than in the monomer form, when solvated by unsaturated lipids. In all lipid environments there is a marked preference for lipids to interact with peptide predominantly through one rather than both acyl chains. Although our trajectories are not long enough to allow a full thermodynamic treatment, these results demonstrate that molecular dynamics simulations are a powerful method for investigating the protein-protein, protein-lipid, and lipid-lipid interactions that determine the structure, stability and dynamics of transmembrane alpha-helices in membranes.

1,2-Dipalmitoylphosphatidylcholine↗

PubMed2000

Manganese selectivity of pmr1, the yeast secretory pathway ion pump, is defined by residue gln783 in transmembrane segment 6. Residue Asp778 is essential for cation transport.

We have solubilized and purified the histidine-tagged yeast secretory pathway/Golgi ion pump Pmr1 to near homogeneity in one step, using nickel affinity chromatography. The purified pump demonstrates both Ca(2+)- and Mn(2+)-dependent ATP hydrolysis and phosphoenzyme intermediate formation in forward (ATP) and reverse (P(i)) directions. This preparation has allowed us to examine, in detail, the properties of mutations D778A and Q783A in transmembrane segment M6 of Pmr1. In phenotypic screens of Ca(2+) chelator and Mn(2+) toxicity reported separately (Wei, Y., Chen, J., Rosas, G., Tompkins, D.A., Holt, P.A., and Rao, R. (2000) J. Biol. Chem. 275, XXXX-XXXX), D778A was a loss-of-function mutant apparently defective for transport of both Ca(2+) and Mn(2+), whereas mutant Q783A displayed a differential sensitivity consistent with the selective loss of Mn(2+) transport. We show that mutant D778A is devoid of cation-dependent ATP hydrolytic activity and phosphoenzyme formation from ATP. However, reverse phosphorylation from P(i) is preserved but is insensitive to inhibition by Ca(2+) or Mn(2+) ions, which is evidence for a specific inability to bind cations in this mutant. We also show that Ca(2+) can activate ATP hydrolysis in the purified Q783A mutant, with a half-maximal concentration of 0.06 micrometer, essentially identical to that of wild type (0.07 micrometer). Mn(2+) activation of ATP hydrolysis was half-maximal at 0.02 micrometer in wild type, establishing a normal selectivity profile of Mn(2+) > Ca(2+). Strikingly, Mn(2+)-ATPase in the Q783A mutant was nearly abolished, even at concentrations of up to 10 micrometer. These results were confirmed in assays of phosphoenzyme intermediates. Molecular modeling of the packing between helices M4 and M6 suggests that residue Gln(783) in M6 may form a critical hydrophobic interaction with Val(335) in M4, such that the Ala substitution modifies the packing or tilt of the helices and thus the ion pore. The data emphasize the critical role of transmembrane segment M6 in defining the cation binding pocket of P-type ATPases.

ATP-Binding Cassette Transporters↗

PubMed2000

Correlation between changes in nuclear magnetic resonance order parameters and conformational entropy: molecular dynamics simulations of native and denatured staphylococcal nuclease.

Recent work has suggested that changes in NMR order parameters may quantitatively reflect changes in the conformational entropy of a protein ensemble. The extent of the mathematical relationship between local entropy changes as seen by NMR order parameters and the full protein entropy change is a complex issue. As a step towards a fuller understanding of this problem, molecular dynamics calculations of both native and denatured staphylococcal nuclease were performed. The N-H bond vector motion, in both explicit and implicit solvent, was analyzed to estimate local and global entropy changes. The calculated N-H bond vector order parameters from simulation agreed on average with experimental values for both native and denatured structures. However, the inverted-U profile of order parameters versus residue number observed experimentally for denatured nuclease was only partially reproduced by simulation of compact denatured structures. Comparisons made across the full set of simulations revealed a correlation between the N-H order parameter-based conformational entropy change and the total quasiharmonic-based conformational entropy change between the native and denatured structures. The calculations showed that about 25% of the total entropy change was reflected by changes in simulated S2 values. This result suggests that NMR-derived order parameters may be used to provide a reasonable estimate of the total conformational entropy change on protein folding.

Magnetic Resonance Spectroscopy↗

PubMed2000

Differences between apo and three holo forms of the intestinal fatty acid binding protein seen by molecular dynamics computer calculations.

It is commonly believed that binding affinity can be estimated by consideration of local changes of ligand and protein. This paper discusses a set of molecular dynamics simulations of intestinal fatty acid binding protein addressing the protein's response to presence or absence of different ligands. A 5-ns simulation was performed of the protein without a ligand, and three simulations (one 5-ns and two 2-ns) were performed with different fatty acids bound. The results indicate that, although the basic protein structure is unchanged by the presence of the ligand, other properties are significantly affected by ligand binding. For example, zero-time covariance patterns between protein, bound waters, and ligand vary between the different simulations. Moreover, the interaction energies between ligand and specific residues indicate that different ligands are stabilized in different ways. In sum, the results suggest that binding thermodynamics within this system will need to be calculated not from a subset of nearby protein:ligand interactions, but will depend on a knowledge of the motions coupling together water, protein, and ligand.

Amino Acids↗

PubMed1999

The binding site of sodium in the gramicidin A channel.

The available information concerning the structure and location of the main binding site for sodium in the gramicidin A channel is reviewed and discussed. Results from molecular dynamics simulations using an atomic model of the channel embedded in a lipid bilayer are compared with experimental observations. The combined information from experiment and simulation suggests that the main binding sites for sodium are near the channel's mouth, approximately 9.2 A from the centre of the dimer channel, although the motion along the axis could be as large as 1 to 2 A. In the binding site, the sodium ion is lying off axis, making contact with two carbonyl oxygens and two single-file water molecules. The main channel ligand is provided by the carbonyl group of the Leu10-Trp11 peptide linkage, which exhibits the largest deflection from the ion-free channel structure.

Anti-Bacterial Agents↗

PubMed1999

The third leg: molecular dynamics simulations of lipid binding proteins.

Molecular dynamics computer simulations can provide a third leg which balances the contributions of both structural biology and binding studies performed on the lipid binding protein family. In this context, these calculations help to establish a dialogue between all three communities, by relating experimental observables with details of structure. Working towards this connection is important, since experience has shown the difficulty of inferring thermodynamic properties from a single static conformation. The challenge is exemplified by ongoing attempts to interpret the impact of mutagenesis on structure and function (i.e. binding). A detailed atomic-level understanding of this system could be achieved with the support of all three legs, paving the way towards rational design of proteins with novel specificities. This paper provides an outline of the connections possible between experiment and theory concerning lipid binding proteins.

Carrier Proteins↗

PubMed1998

Simulations of fatty acid-binding proteins suggest sites important for function. I. Stearic acid.

Molecular dynamics simulations of two structurally similar fatty acid-binding proteins interacting with stearic acid are described. The calculations relate to recent ligand binding measurements and suggest similarities and differences between the two systems. Charged and neutral forms of the fatty acid were examined. The charged forms led to rapid trajectory divergence, whereas the protonated forms remained stable over the length of their 1-ns production trajectories. The two protein systems showed similar sets of total interaction energies with the ligand. However, the strengths of individual amino acids interacting with the ligand differ. Furthermore, covariance analysis of the ligand with both protein and water suggests that the stearic acid in the adipocyte fatty acid-binding protein is coupled more strongly to the water than to the protein. The stearic acid in the muscle fatty acid-binding protein is seen to be coupled differentially along the length of the chain to the protein. These differences could help to rationalize the stronger binding affinity for stearic acid in the human muscle fatty acid-binding protein. An importance scale, based on both covariance and interaction energy with the ligand, is proposed to identify residues that may be important for binding function.

Adipocytes↗

PubMed1998

Simulations of fatty acid-binding proteins. II. Sites for discrimination of monounsaturated ligands.

Fatty acid binding proteins (FABPs) can discriminate between saturated and unsaturated fatty acids via molecular mechanisms that are not understood. Molecular dynamics computer calculations are used to suggest the relationship between tertiary structure and binding specificity. Three separate 1-ns simulations, with explicit solvent, are presented: 1) oleic acid (C18:1 cis) bound to adipocyte FABP, 2) oleic acid bound to human muscle FABP, and 3) elaidic acid (C18:1 trans) bound to human muscle FABP. The average structural, dynamic, and energetic properties of the trajectory were analyzed, as were the motional correlations. The molecular dynamics trajectories reveal intriguing differences among all three systems. For example, the two proteins have different strengths of interaction energy with the ligand and different motional coupling, as seen with covariance analysis. This suggests distinctive molecular behavior of monounsaturated fatty acids in the two similar proteins. An importance scale, based on motional correlation and interaction energy between protein and ligand, is proposed, to help identify amino acids involved with the discrimination of ligand saturation state or geometric isomerization.

Adipocytes↗

PubMed1998

Molecular dynamics simulations of individual alpha-helices of bacteriorhodopsin in dimyristoylphosphatidylcholine. II. Interaction energy analysis.

The concepts of hydrophobicity and hydrophobic moments have been applied in attempts to predict membrane protein secondary and tertiary structure. The current paper uses molecular dynamics computer calculations of individual bacteriorhodopsin helices in explicit dimyristoylphosphatidylcholine bilayers to examine the atomic basis of these approaches. The results suggest that the types of interactions between a particular amino acid and the surrounding bilayer depend on the position and type of the amino acid. In particular, aromatic residues are seen to interact favorably at the interface region. Analysis of the trajectories in terms of hydrophobic moments suggests the presence of a particular face that prefers lipid. The results of these simulations may be used to improve secondary structure prediction methods and to provide further insights into the two-stage model of protein folding.

Amino Acids↗

PubMed1997

Molecular dynamics of individual alpha-helices of bacteriorhodopsin in dimyristol phosphatidylocholine. I. Structure and dynamics.

Understanding the role of the lipid bilayer in membrane protein structure and dynamics is needed for tertiary structure determination methods. However, the molecular details are not well understood. Molecular dynamics computer calculations can provide insight into these molecular details of protein:lipid interactions. This paper reports on 10 simulations of individual alpha-helices in explicit lipid bilayers. The 10 helices were selected from the bacteriorhodopsin structure as representative alpha-helical membrane folding components. The bilayer is constructed of dimyristoyl phosphatidylcholine molecules. The only major difference between simulations is the primary sequence of the alpha-helix. The results show dramatic differences in motional behavior between alpha-helices. For example, helix A has much smaller root-mean-squared deviations than does helix D. This can be understood in terms of the presence of aromatic residues at the interface for helix A that are not present in helix D. Additional motions are possible for the helices that contain proline side chains relative to other amino acids. The results thus provide insight into the types of motion and the average structures possible for helices within the bilayer setting and demonstrate the strength of molecular simulations in providing molecular details that are not directly visualized in experiments.

Amino Acid Sequence↗

PubMed1997

Structure and dynamics of an amphiphilic peptide in a lipid bilayer: a molecular dynamics study.

A molecular dynamics simulation of a simple model membrane system composed of a single amphiphilic helical peptide (ace-K2GL16K2A-amide) in a fully hydrated 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer was performed for a total of 1060 ps. The secondary structure of the peptide and its stability were described in terms of average dihedral angles, phi and psi, and the C alpha torsion angles formed by backbone atoms; by the average translation per residue along the helix axis; and by the intramolecular peptide hydrogen bonds. The results indicated that residues 6 through 15 remain in a stable right-handed alpha-helical conformation, whereas both termini exhibit substantial fluctuations. A change in the backbone dihedral angles for residues 16 and 17 is accompanied by the loss of two intramolecular hydrogen bonds, leading to a local but long-lived disruption of the helix. The dynamics of the peptide was characterized in terms of local and global helix motions. The local motions of the N-H bond angles were described in terms of the autocorrelation functions of P2[cos thetaNH(t, t + tau)] and reflected the different degrees of local peptide order as well as a variation in time scale for local motions. The chi1 and chi2 dihedral angles of the leucine side chains underwent frequent transitions between potential minima. No connection between the side-chain positions and their mobility was observed, however. In contrast, the lysine side chains displayed little mobility during the simulation. The global peptide motions were characterized by the tilting and bending motions of the helix. Although the peptide was initially aligned parallel to the bilayer normal, during the simulation it was observed to tilt away from the normal, reaching an angle of approximately 25 degrees by the end of the simulation. In addition, a slight bend of the helix was detected. Finally, the solvation of the peptide backbone and side-chain atoms was also investigated.

Amino Acid Sequence↗

PubMed1997

The binding site of sodium in the gramicidin A channel: comparison of molecular dynamics with solid-state NMR data.

The location of the main binding site for sodium in the gramicidin A (GA) channel was investigated with molecular dynamics simulations, using an atomic model of the channel embedded in a fully hydrated dimyristoyl phosphatidycholine (DMPC) bilayer. Twenty-four separate simulations in which a sodium was restrained at different locations along the channel axis were generated. The results are compared with carbonyl 13C chemical shift anisotropy solid-state NMR experimental data previously obtained with oriented GA:DMPC samples. Predictions are made for other solid-state NMR properties that could be observed experimentally. The combined information from experiment and simulation strongly suggests that the main binding sites for sodium are near the channel's mouth, approximately 9.2 A from the center of the dimer channel. The 13C chemical shift anisotropy of Leu10 is the most affected by the presence of a sodium ion in the binding site. In the binding site, the sodium ion is lying off-axis, making contact with two carbonyl oxygens and two single-file water molecules. The main channel ligand is provided by the carbonyl group of the Leu10-Trp11 peptide linkage, which exhibits the largest deviation from the ion-free channel structure. Transient contacts with the carbonyl group of Val8 and Trp15 are also present. The influence of the tryptophan side chains on the channel conductance is examined based on the current information about the binding site.

Binding Sites↗

PubMed1996

Structure, energetics, and dynamics of lipid-protein interactions: A molecular dynamics study of the gramicidin A channel in a DMPC bilayer.

The microscopic details of lipid-protein interactions are examined using molecular dynamics simulations of the gramicidin A channel embedded in a fully hydrated dimyristoyl phosphatidylcholine (DMPC) bilayer. A novel construction protocol was used to assemble the initial configurations of the membrane protein complex for the simulations. Three hundred systems were constructed with different initial lipid placement and conformations. Seven systems were simulated with molecular dynamics. One system was simulated for a total of 600 psec, four were simulated for 300 psec, and two for 100 psec. Analysis of the resulting trajectories shows that the bulk solvent-membrane interface region is much broader than traditionally pictured in simplified continuum theories: its width is almost 15 angstroms. In addition, lipid-protein interactions are far more varied, both structurally and energetically, than is usually assumed: the total interaction energy between the gramicidin A and the individual lipids varies from 0 to -50 kcal/mol. The deuterium quadrupolar splittings of the lipid acyl chains calculated from the trajectories are in good agreement with experimental data. The lipid chains in direct contact with the GA are ordered but the effect is not uniform due to the irregular surface of the protein. Energy decompositions shows that the most energetically favorable interactions between lipid and protein involve nearly equal contributions from van der Waals and electrostatic interactions. The tryptophans, located near the bulk-membrane interface, appear to be particularly important in mediating both hydrogen bonding interactions with the lipid glycerol backbone and water and also in forming favorable van der Waals contacts with the hydrocarbon chains. In contrast, the interactions of the leucine residues with the lipids, also located near the interface, are dominated by van der Waals interactions with the hydrocarbon lipid chains.

Biophysics↗

PubMed1994

Molecular dynamics simulation of the gramicidin channel in a phospholipid bilayer.

A molecular dynamics simulation of the gramicidin A channel in an explicit dimyristoyl phosphatidylcholine bilayer was generated to study the details of lipid-protein interactions at the microscopic level. Solid-state NMR properties of the channel averaged over the 500-psec trajectory are in excellent agreement with available experimental data. In contrast with the assumptions of macroscopic models, the membrane/solution interface region is found to be at least 12 A thick. The tryptophan side chains, located within the interface, are found to form hydrogen bonds with the ester carbonyl groups of the lipids and with water, suggesting their important contribution to the stability of membrane proteins. Individual lipid-protein interactions are seen to vary from near 0 to -50 kcal/mol. The most strongly interacting conformations are short-lived and have a nearly equal contribution from both van der Waals and electrostatic energies. This approach for performing molecular dynamics simulations of membrane proteins in explicit phospholipid bilayers should help in studying the structure, dynamics, and energetics of lipid-protein interactions.

Gramicidin↗

PubMed1994

Local communication within dendritic spines: models of second messenger diffusion in granule cell spines of the mammalian olfactory bulb.

Dendritic spines are generally believed to play a role in modulating synaptically induced electrical events. In addition, they may also confine second messengers and thus topologically limit the distance over which second messenger cascades may be functionally significant. In order to address this possibility, computer simulations of transient second messenger concentration changes were performed. The results show the importance of spine morphology and binding and extrusion mechanisms in controlling second messenger transients. In the presence of intrinsic cytoplasmic binding sites and kinetic rates similar to that expected for calcium, second messengers were confined to the spine head. In the absence of binding/extrusion mechanisms, the size and time course of the input transient to the spine head influenced the second messenger transients that might be seen at the base of the spine neck and in other spines. Large and/or sustained increases in second messenger concentration in the spine head were communicated to the spine base and to other spine heads. The results emphasize the importance of a knowledge of breakdown pathways, concentrations and kinetics of binding sites, and extrusion mechanisms for understanding the dynamics of local chemical changes for dendritic spine function.

Animals↗