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Kim A Sharp

Publications and source records attributed to Kim A Sharp.

16 recordsLinked to original sources
PubMed2007

Temperature dependence of fast dynamics in proteins.

The temperature dependence of the internal dynamics of recombinant human ubiquitin has been measured using solution NMR relaxation techniques. Nitrogen-15 relaxation has been employed to obtain a measure of the amplitude of subnanosecond motion at amide N-H sites in the protein. Deuterium relaxation has been used to obtain a measure of the amplitude of motion of methyl-groups in amino-acid side chains. Data was obtained between 5 and 55 degrees C. The majority of amide N-H and methyl groups show a roughly linear (R(2)>0.75) temperature dependence of the associated Lipari-Szabo model-free squared generalized-order parameter (O(2)) describing the amplitude of motion. Interestingly, for those sites showing a linear response, the temperature dependence of the backbone is distinct from that of the methyl-bearing side chains with the former being characterized by a significantly larger Lambda-value, where Lambda is defined as d ln(1 - O)/d lnT. These results are comparable to the sole previous such study of the temperature dependence of protein motion obtained for a calmodulin-peptide complex. This suggests that the distinction between the main chain and methyl-bearing side chains may be general. Insight into the temperature dependence is gathered from a simple two-state step potential model.

Computer Simulation↗
PubMed2006

Travel depth, a new shape descriptor for macromolecules: application to ligand binding.

Depth is a term frequently applied to the shape and surface of macromolecules, describing for example the grooves in DNA, the shape of an enzyme active site, or the binding site for a small molecule in a protein. Yet depth is a difficult property to define rigorously in a macromolecule, and few computational tools exist to quantify this notion, to visualize it, or analyze the results. We present our notion of travel depth, simply put the physical distance a solvent molecule would have to travel from a surface point to a suitably defined reference surface. To define the reference surface, we use the limiting form of the molecular surface with increasing probe size: the convex hull. We then present a fast, robust approximation algorithm to compute travel depth to every surface point. The travel depth is useful because it works for pockets of any size and complexity. It also works for two interesting special cases. First, it works on the grooves in DNA, which are unbounded in one direction. Second, it works on the case of tunnels, that is pockets that have no "bottom", but go through the entire macromolecule. Our algorithm makes it straightforward to quantify discussions of depth when analyzing structures. High-throughput analysis of macromolecule depth is also enabled by our algorithm. This is demonstrated by analyzing a database of protein-small molecule binding pockets, and the distribution of bound magnesium ions in RNA structures. These analyses show significant, but subtle effects of depth on ligand binding localization and strength.

Algorithms↗
PubMed2006

Hydrogen bonding and the cryoprotective properties of glycerol/water mixtures.

Molecular dynamics simulations and infrared spectroscopy were used to determine the hydrogen bond patterns of glycerol and its mixtures with water. The ability of glycerol/water mixtures to inhibit ice crystallization is linked to the concentration of glycerol and the hydrogen bonding patterns formed by these solutions. At low glycerol concentrations, sufficient amounts of bulk-like water exist, and at low temperature, these solutions demonstrate crystallization. As the glycerol concentration is increased, the bulk-like water pool is eventually depleted. Water in the first hydration shell becomes concentrated around the polar groups of glycerol, and the alkyl groups of glycerol self-associate. Glycerol-glycerol hydrogen bonds become the dominant interaction in the first hydration shell, and the percolation nature of the water network is disturbed. At glycerol concentrations beyond this point, glycerol/water mixtures remain glassy at low temperatures and the glycerol-water hydrogen bond favors a more linear arrangement. High glycerol concentration mixtures mimic the strong hydrogen bonding pattern seen in ice, yet crystallization does not occur. Hydrogen bond patterns are discussed in terms of hydrogen bond angle distributions and average hydrogen bond number. Shift in infrared frequency of related stretch and bend modes is also reviewed.

Cryoprotective Agents↗
PubMed2005

Carbohydrate intramolecular hydrogen bonding cooperativity and its effect on water structure.

Molecular dynamics (MD) simulations combined with water-water H-bond angle analysis and calculation of solvent accessible surface area and approximate free energy of solvation were used to determine the influence of hydroxyl orientation on solute hydration and surrounding water structure for a group of chemically identical solutes-the aldohexopyranose sugars. Intramolecular hydrogen bond cooperativity was closely associated with changes in water structure surrounding the aldohexopyranose stereoisomers. The OH-4 group played a pivotal role in hydration as it was able to participate in a number of hydrogen bond networks utilizing the OH-6 group. Networks that terminated within the molecule (OH-4 --> OH-6 --> O-5) had relatively more nonpolar-like hydration than those that ended in a free hydroxyl group (OH-6 --> OH-4 --> OH-3). The OH-2 group modulated the strength of OH-4 networks through syndiaxial OH-2/4 intramolecular hydrogen bonding, which stabilized and induced directionality in the network. Other syndiaxial interactions, such as the one between OH-1 and OH-3, only indirectly affected water structure. Water structure surrounding hydrogen bond networks is discussed in terms of water-water hydrogen bond populations. The impact of syndiaxial versus vicinal hydrogen bonds is also reviewed. The results suggest that biological events such as protein-carbohydrate recognition and cryoprotection by carbohydrates may be driven by intramolecular hydrogen bond cooperativity.

Carbohydrates↗
PubMed2005

Hydrophobic tendency of polar group hydration as a major force in type I antifreeze protein recognition.

The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of distortions of the water-water hydrogen bonding angle in the first hydration shell (Gallagher and Sharp, JACS 2003;125:9853). The distribution of this angle in pure water is bimodal, with a low-angle population and high-angle population. Polar solutes increase the high-angle population while apolar solutes increase the low-angle population. The ratio of the two populations quantifies the hydrophobicity of the solute and provides a sensitive measure of water structural distortions. This method of analysis is applied to study hydration of type I thermal hysteresis protein (THP) from winter flounder and three quadruple mutants of four threonine residues at positions 2, 13, 24, and 35. Wild-type and two mutants (VVVV and AAAA) have antifreeze (thermal hysteresis) activity, while the other mutant (SSSS) has no activity. The analysis reveals significant differences in the hydration structure of the ice-binding site. For the SSSS mutant, polar groups have a typical polar-like hydration, that is, more high-angle H-bonds than bulk water. For the wild-type and active mutants, polar groups have unusual, very apolar-like hydration, that is, more low-angle H-bonds than bulk water. This pattern of hydration was seen previously in the structurally distinct type III THPs (Yang & Sharp Biophys Chem 2004;109:137), suggesting for the first time a general mechanism for different THP classes. The specific shape, residue size, and clustering of both polar and apoler groups are essential for an active ice binding surface.

Antifreeze Proteins, Type I↗
PubMed2005

Heat capacity in proteins.

Heat capacity (Cp) is one of several major thermodynamic quantities commonly measured in proteins. With more than half a dozen definitions, it is the hardest of these quantities to understand in physical terms, but the richest in insight. There are many ramifications of observed Cp changes: The sign distinguishes apolar from polar solvation. It imparts a temperature (T) dependence to entropy and enthalpy that may change their signs and which of them dominate. Protein unfolding usually has a positive deltaCp, producing a maximum in stability and sometimes cold denaturation. There are two heat capacity contributions, from hydration and protein-protein interactions; which dominates in folding and binding is an open question. Theoretical work to date has dealt mostly with the hydration term and can account, at least semiquantitatively, for the major Cp-related features: the positive and negative Cp of hydration for apolar and polar groups, respectively; the convergence of apolar group hydration entropy at T approximately 112 degrees C; the decrease in apolar hydration Cp with increasing T; and the T-maximum in protein stability and cold denaturation.

Cold Temperature↗
PubMed2004

The mechanism of the type III antifreeze protein action: a computational study.

The random network model of water quantitatively describes the different hydration heat capacities of polar and apolar solutes in terms of differential distortions of the water-water hydrogen bonding angle in the first hydration shell. This method of hydration analysis is applied here to study the hydration of the wild type III thermal hysteresis protein from eel pout and three mutations at residue 16. Wild type and one mutant have full activity, the other two mutants have little or no anti-freeze (thermal hysteresis) activity. The analysis reveals significant differences in the hydration structure of the ice-binding site (centered on residue 16) among four proteins. For the A16T and A16Y mutants with reduced activity, polar groups have a typical polar-like hydration. For the wild type and mutant A16C with 100% of the wild type activity, polar groups have unusual, very apolar-like hydration. In the latter case, hydrating water molecules form a more ice-like pattern of hydrogen bonding on the ice-binding face, while in the former case water-water H-bonds are more distorted and more heterogenous. Overall, the binding surface of active protein strongly enhances the water tetrahedral structure, i.e. promotes ice-like hydration. It is concluded that the specific shape, residue size and clustering of both polar/apolar groups are essential for the binding surface to recognize, and preferentially interact with nascent ice crystals forming in liquid water.

Antifreeze Proteins, Type III↗
PubMed2004

Implementation and testing of stable, fast implicit solvation in molecular dynamics using the smooth-permittivity finite difference Poisson-Boltzmann method.

A fast stable finite difference Poisson-Boltzmann (FDPB) model for implicit solvation in molecular dynamics simulations was developed using the smooth permittivity FDPB method implemented in the OpenEye ZAP libraries. This was interfaced with two widely used molecular dynamics packages, AMBER and CHARMM. Using the CHARMM-ZAP software combination, the implicit solvent model was tested on eight proteins differing in size, structure, and cofactors: calmodulin, horseradish peroxidase (with and without substrate analogue bound), lipid carrier protein, flavodoxin, ubiquitin, cytochrome c, and a de novo designed 3-helix bundle. The stability and accuracy of the implicit solvent simulations was assessed by examining root-mean-squared deviations from crystal structure. This measure was compared with that of a standard explicit water solvent model. In addition we compared experimental and calculated NMR order parameters to obtain a residue level assessment of the accuracy of MD-ZAP for simulating dynamic quantities. Overall, the agreement of the implicit solvent model with experiment was as good as that of explicit water simulations. The implicit solvent method was up to eight times faster than the explicit water simulations, and approximately four times slower than a vacuum simulation (i.e., with no solvent treatment).

Algorithms↗
PubMed2003

Accessibility of oxygen with respect to the heme pocket in horseradish peroxidase.

Oxygen and other molecules of similar size take part in a variety of protein reactions. Therefore, it is critical to understand how these small molecules penetrate the protein matrix. The protein system studied in this case is horseradish peroxidase (HRP). We have converted the native HRP into a phosphorescent analog by replacing the heme prosthetic group by Pd-mesoporphyrin. Oxygen readily quenches the phosphorescence of Pd porphyrins, and this can be used to characterize oxygen diffusion through the protein matrix. Our measurements indicate that solvent viscosity and pH modulate the accessibility of the heme pocket relative to small molecules. The binding of the substrate benzohydroxamic acid (BHA) to the protein drastically impedes oxygen access to the heme pocket. These results indicate that, first, the penetration of small molecules through the protein matrix is a function of protein dynamics, and second, there are specific pathways for the diffusion of these molecules. The effect of substrate and pH on protein dynamics has been investigated with the use of molecular dynamics calculations. We demonstrate that the model of a "fluctuating entry point," as suggested by Zwanzig (J Chem Phys 1992;97:3587-3589), properly describes the diffusion of oxygen through the protein matrix.

Binding Sites↗
PubMed2003

Solvent dependent and independent motions of CO-horseradish peroxidase examined by infrared spectroscopy and molecular dynamics calculations.

The role of the solvent matrix in affecting CO bound to ferrous horseradish peroxidase was examined by comparing band-widths of nu(CO) for the protein in aqueous solutions and in trehalose/sucrose glasses. We have previously observed that the optical absorption band and the CO stretching mode respond to the glass transition of glycerol/water in ways that depend upon the presence of substrate (Biochemistry 40 (2001) 3483). It is now demonstrated that the CO group band-width for the protein with bound inhibitor benzhydroxamic acid is relatively insensitive to temperature or the glass transition of the solvent. In contrast, in the absence of inhibitor, the band-width varies with the temperature that the glass is formed. The results show that solvent dependent and independent motions can be distinguished, and that the presence of substrate changes the protein such that the Fe[bond]CO site is occluded from the solvent conditions. Molecular dynamic calculations, based upon X-ray structures, showed that the presence of benzhydroxamic acid decreases the distance between His42 and Arg38 and this leads for closer distances to the O of the CO from these residues. These results are invoked to account for the observed line width changes of the CO band.

Arginine↗
PubMed2003

A new angle on heat capacity changes in hydrophobic solvation.

The differential solubility of polar and apolar groups in water is important for the self-assembly of globular proteins, lipid membranes, nucleic acids, and other specific biological structures through hydrophobic and hydrophilic effects. The increase in water's heat capacity upon hydration of apolar compounds is one signature of the hydrophobic effect and differentiates it from the hydration of polar compounds, which cause a decrease in heat capacity. Water structuring around apolar and polar groups is an important factor in their differential solubility and heat capacity effects. Here, it is shown that joint radial/angular distribution functions of water obtained from simulations reveal quite different hydration structures around polar and apolar groups: polar and apolar groups have a deficit or excess, respectively, of "low angle hydrogen bonds". Low angle hydrogen bonds have a larger energy fluctuation than high angle bonds, and analysis of these differences provides a physical reason for the opposite changes in heat capacity and new insight into water structure around solutes and the hydrophobic effect.

Computer Simulation↗
PubMed2003

Dynamics and entropy of a calmodulin-peptide complex studied by NMR and molecular dynamics.

All-atom, explicit water molecular dynamics simulations of calcium-loaded calmodulin complexed with a peptide corresponding to the smooth muscle myosin light chain kinase target were carried out at 295 and 346 K. Amide and side chain methyl angular generalized order parameters were calculated and analyzed in the context of the protein's structure and dynamics. The agreement between amide order parameters measured by NMR and those from the simulations was found to be good, especially at the higher temperature, indicating both better convergence for the latter and excellent transferrability of the CHARMM parameters to the higher temperature. Subtle dynamical features such as helix fraying were reproduced. A large range of order parameters for the nine calmodulin methionines was observed in the NMR, and reproduced quite well in the simulations. The major determinant of the methionine order parameter was found to be the proximity to side chains of aromatic residues. An upper bound estimate of the difference in backbone entropy between loop and helical regions was extracted from the order parameters using a model of motion in an effective potential. Although loop regions are more flexible than helical regions, it was found that the entropy loss per residue upon folding was only approximately 20% less for loops than for helices. Pairwise correlated motions, which could significantly lower entropy estimates obtained from order parameter analysis alone, were found to be largely absent.

Amides↗
PubMed2003

Analysis of thermal hysteresis protein hydration using the random network model.

The hydration of polar and apolar groups can be explained quantitatively, via the random network model of water, in terms of differential distortions in first hydration shell water-water hydrogen bonding angle. This method of analyzing solute induced structural distortions of water is applied to study the ice-binding type III thermal hysteresis protein. The analysis reveals subtle but significant differences in solvent structuring of the ice-binding surface, compared to non-ice binding protein surface. The major differences in hydration in the ice-binding region are (i). polar groups have a very apolar-like hydration. (ii). there is more uniform hydration structure. Overall, this surface strongly enhances the tetrahedral, or ice-like, hydration within the primary hydration shell. It is concluded that these two specific features of the hydration structure are important for this surface to recognize, and preferentially interact with nascent ice crystals forming in liquid water.

Animals↗
PubMed2002

Temperature dependence of the internal dynamics of a calmodulin-peptide complex.

The temperature dependence of the fast internal dynamics of calcium-saturated calmodulin in complex with a peptide corresponding to the calmodulin-binding domain of the smooth muscle myosin light chain kinase is examined using 15N and 2H NMR relaxation methods. NMR relaxation studies of the complex were carried out at 13 temperatures that span 288-346 K. The dynamics of the backbone and over four dozen methyl-bearing side chains, distributed throughout the calmodulin molecule, were probed. The side chains show a much more variable and often considerably larger response to temperature than the backbone. A significant variation in the temperature dependence of the amplitude of motion of individual side chains is seen. The amplitude of motion of some side chains is essentially temperature-independent while many show a simple roughly linear temperature dependence. In a few cases, angular order increases with temperature, which is interpreted as arising from interactions with neighboring residues. In addition, a number of side chains display a nonlinear temperature dependence. The significance of these and other results is illuminated by several simple interpretative models. Importantly, analysis of these models indicates that changes in generalized order parameters can be robustly related to corresponding changes in residual entropy. A simple cluster model that incorporates features of cooperative or conditional motion reproduces many of the unusual features of the experimentally observed temperature dependence and illustrates that side chain interactions result in a dynamically changing environment that significantly influences the motion of internal side chains. This model also suggests that the intrinsic entropy of interacting clusters of side chains is only modestly reduced from that of independent side chain motion. Finally, estimates of protein heat capacity support the view that the major contribution to the heat capacity of protein solutions largely arises from local bond vibrations and solvent interactions and not from torsional oscillations of side chains.

Animals↗
PubMed2002

Stability of macromolecular complexes.

Macromolecular interactions are crucial in numerous biologic processes, yet few general principles are available that establish firm expectations for the strength of these interactions or the expected contribution of specific forces. The simplest principle would be a monotonic increase in interactions as the size of the interface grows. The exact relationship might be linear or nonlinear depending on the nature of the forces involved. Simple "linear-free energy" relationships based on atomic properties have been well documented, for example, additivity for the interaction of small molecules with solvent, and, recently, have been explored for ligand-receptor interactions. Horton and Lewis propose such additivity based on buried surface area for protein-protein complexes. We investigated macromolecular interactions and found that the highest-affinity complexes do not fulfill this simple expectation. Instead, binding free energies of the tightest macromolecular complexes are roughly constant, independent of interface size, with the notable exception of DNA duplexes. By comparing these results to an earlier study of protein-ligand interactions we find that: (1) The maximum affinity is approximately 1.5 kcal/mol per nonhydrogen atom or 120 cal/mol A(2) of buried surface area, comparable to results of our earlier work; (2) the lack of an increase in affinity with interface size is likely due to nonthermodynamic factors, such as functional and evolutionary constraints rather than some fundamental physical limitation. The implication of these results have some importance for molecular design because they suggest that: (1) The stability of any given complex can be increased significantly if desired; (2) small molecule inhibitors of macromolecular interactions are feasible; and (3) different functional classes of protein-protein complexes exhibit differences in maximal stability, perhaps in response to differing evolutionary pressures. These results are consistent with the widespread observation that proteins have not evolved to maximize thermodynamic stability, but are only marginally stable.

Animals↗
PubMed2002

Structural determination of spin label immobilization and orientation: a Monte Carlo minimization approach.

Electron paramagnetic resonance (EPR) is often used in the study of the orientation and dynamics of proteins. However, there are two major obstacles in the interpretation of EPR signals: (a) most spin labels are not fully immobilized by the protein, hence it is difficult to distinguish the mobility of the label with respect to the protein from the reorientation of the protein itself; (b) even in cases where the label is fully immobilized its orientation with respect to the protein is not known, which prevents interpretation of probe reorientation in terms of protein reorientation. We have developed a computational strategy for determining whether or not a spin label is immobilized and, if immobilized, predicting its conformation within the protein. The method uses a Monte Carlo minimization algorithm to search the conformational space of labels within known atomic level structures of proteins. To validate the method a series of spin labels of varying size and geometry were docked to sites on the myosin head catalytic and regulatory domains. The predicted immobilization and conformation compared well with the experimentally determined mobility and orientation of the label. Thus, probes can now be targeted to report on various modes of molecular dynamics: immobilized probes to report on protein backbone and domain dynamics or floppy probes to report on the extent of steric restriction experienced by the side chain.

Animals↗