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K A Sharp

Publications and source records attributed to K A Sharp.

33 records · Page 2Linked to original sources
PubMed1994

Salt effects on protein-DNA interactions. The lambda cI repressor and EcoRI endonuclease.

In this paper, finite-difference solutions to the nonlinear Poisson-Boltzmann (NLPB) equation are used to calculate the salt dependent contribution to the electrostatic DNA binding free energy for both the lambda cI repressor and the EcoRI endonuclease. For the protein-DNA systems studied, the NLPB method describes nonspecific univalent salt dependent effects on the binding free energy which are in excellent agreement with experimental results. In these systems, the contribution of the ion atmosphere to the binding free energy substantially destabilizes the protein-DNA complexes. The magnitude of this effect involves a macromolecular structure dependent redistribution of both cations and anions around the protein and the DNA which is dominated by long range electrostatic interactions. We find that the free energy associated with global ion redistribution upon binding is more important than changes associated with local protein-DNA interactions (ion-pairs) in determining salt effects. The NLPB model reveals how long range salt effects can play a significant role in the relative stability of protein-DNA complexes with different structures.

Bacteriophage lambda↗
PubMed1994

Electric field and conformational effects of cytochrome c and solvent on cytochrome c peroxidase studied by high-resolution fluorescence spectroscopy.

Electronic spectra of mesoporphyrin-substituted yeast cytochrome c peroxidase (MP-CcP) were measured as a function of pH, ionic strength, and binding of cytochrome c (cyt c) by fluorescence line narrowing (FLN) spectroscopy at 5 K. The FLN spectra provided information about the vibrational structure of the first excited singlet state of MP-CcP, the various tautomeric forms of mesoporphyrin, and the positions and widths of their 0,0 bands. The composite 0,0 band of MP-CcP at pH 6 could be resolved into three components with peak positions at 16,046, 16,103, and 16,203 cm-1. MP-CcP at pH 8 could be analyzed using two components with peak positions at 16,048 and 16,193 cm-1. The disappearance of the 16,103-cm-1 component at alkaline pH suggests that it is due to a "chemical substate" arising from protonation of His52 in the distal side of the porphyrin. Computer simulations of the electrostatic field that CcP imposes on its porphyrin show that, in the presence of charged axial histidines His52 and His175, the electrostatic field at porphyrin nitrogens increases, especially along the normal to the heme by about 200 mV/A. Electric field effects may account for pH-dependent spectral shifts of the 0,0 positions of the resolved components, although hydrogen bonding may also affect these positions. On the other hand, the peak position of the components was not affected by ionic strength or binding of cyt c, implying that the electrostatic field of the heme pocket of MP-CcP remains unchanged. Indeed, computed changes in ionic strength of the solvent show no modification of the electrostatic field at the porphyrin. The only detectable effect of ionic strength and binding of cyt c to MP-CcP is on the relative contributions of the components, suggesting some rearrangements in the vicinity of the heme. Finally, shifts in the position of the vibrational lines for MP-CcP components indicate either that the tautomers have different vibrational frequencies due to the nonsymmetry of the porphyrin and/or that tautomers experience various distortions. Comparison of the vibrational spectrum of the first excited singlet state of mesoporphyrin in CcP and horseradish peroxidase also suggests that the heme pocket in the two peroxidases provides different steric restrictions.

Binding Sites↗
PubMed1994

Correlating solvation free energies and surface tensions of hydrocarbon solutes.

A simple equation relating ratios of transfer free energies and solvation free energies to surface tension is derived. When applied to hydrocarbons in water, experimental values for macroscopic surface tension yield remarkably accurate predictions of a ratio involving microscopic quantities, if one uses transfer free energies which have been adjusted for the effects of solute/solvent volume differences. The results support the validity of applying macroscopic concepts such as interfacial free energy at the molecular level. They further suggest that molecular volume as well as surface area contributes to the solubility of hydrocarbons in water.

Alkanes↗
PubMed1994

How much is a stabilizing bond worth?

It is commonly supposed that the contribution of a bond to protein or nucleic acid stability is equal to the in situ stability of the bond itself. This is not true for the noncovalent bonds that stabilize molecular folding. In general, a bonding interaction contributes a free energy increment to protein or nucleic acid stability that is larger, an enthalpy increment that is smaller, and entropy and heat capacity increments that are more positive than the corresponding bond parameter.

Drug Stability↗
PubMed1994

Calculation of electrostatic effects at the amino terminus of an alpha helix.

It is generally believed that the electrostatic field arising from the dipolar charge distribution in alpha helices is important for protein structure and function. We report a calculation of the electrostatic potential and field at the amino terminus of an alpha helix in water, obtained from a finite difference solution to the Poisson-Boltzmann equation. This method takes into account the detailed helix shape and charge distribution, as well as solvent, and generalized ionic strength effects. The calculated potential and field are found to be in good agreement with the experimentally observed helix-induced Stark effect and pKa shifts of a probe at the N-terminus of a stable, monomeric alpha-helical peptide (Lockhart and Kim, 1992, 1993). Ionic screening effects are reproduced at low salt concentrations. Deviations at higher salt concentrations may result from specific ion effects (specific ion-solute and/or ion-solvent interactions). The FDPB method was used to analyze the contributions from each residue, charged side chains, and solvent to the helix potential and field. Backbone contributions come primarily from the first one to two helical turns. Charged side chains contribute to helix-induced pKa shifts for certain probe-peptide combinations, even at relatively large distances from the probe (> 14 A).

Amino Acid Sequence↗
PubMed1992

Analysis of the heat capacity dependence of protein folding.

This paper presents an analysis of plots of enthalpy versus heat capacity change at 25 degrees C for the unfolding of proteins and for the dissolution of gaseous, liquid and solid solutes, first reported by Murphy, Privalov & Gill. The negative slope in the enthalpy plot for proteins is interpreted as arising from a large penalty associated with burying polar groups in the protein interior. The small enthalpy changes that accompany protein unfolding at 25 degrees C are also discussed. It is argued that the combined effects of hydrogen bond formation and close packing predict a large positive enthalpy of unfolding. Electrostatic calculations indicate that the penalty associated with burying polar groups is large enough to effectively cancel these terms, leading to the small net enthalpy changes that are observed. The free energy changes associated with protein folding are also discussed. The free energy cost of burying polar groups largely compensates for the stabilizing contribution of the hydrophobic effect and would appear to account for the fact that proteins are marginally stable, independent of their size and of their relative hydrophobicities.

Hydrogen Bonding↗
PubMed1991

Extracting hydrophobic free energies from experimental data: relationship to protein folding and theoretical models.

Solubility and vapor pressure measurements of hydrocarbons in water are generally thought to provide estimates of the strength of the hydrophobic effect in the range 20-30 cal/(mol.A2). Our reassessment of the solubility data on the basis of new developments in solution thermodynamics suggests that the hydrophobic surface free energy for hydrocarbon solutes is 46-47 cal/(mol.A2), although the actual value depends strongly on curvature effects [Nicholls et al. (1991) Proteins (in press); Sharp et al. (1991) Science 252, 106-109]. The arguments to support such a significant increase in the estimate of the hydrophobic effect stem partly from theoretical considerations and partly from the experimental results of De Young and Dill [(1990) J. Phys. Chem. 94, 801-809] on benzene partition between water and alkane solvents. Previous estimates of the hydrophobic effect derive from an analysis of solute partition data, which does not fully account for changes in volume entropy. We show here how the ideal gas equations, combined with experimental molar volumes, can account for such changes. Revised solubility scales for the 20 amino acids, based on cyclohexane to water and octanol to water transfer energies, are derived. The agreement between these scales, particularly the octanol scale, and mutant protein stability measurements from Kellis et al. [(1989) Biochemistry 28, 4914-4922] and Shortle et al. [(1990) Biochemistry 29, 8033-8041] is good. The increased strength of the hydrophobic interaction has implications for the energetics of protein folding, substrate binding, and nucleic acid base stacking and the interpretation of computer simulations.

Alkanes↗
PubMed1991

Linkage of thioredoxin stability to titration of ionizable groups with perturbed pKa.

The highly conserved, buried, Asp 26 in Escherichia coli thioredoxin has a pKa = 7.5, and its titration is associated with a sizable destabilization of the protein [Langsetmo, K., Fuchs, J., & Woodward, C. (1991) Biochemistry (preceding paper in this issue)]. A fit of the experimental pH dependence of thioredoxin stability to a theoretical expression for the pH/stability relation in proteins agrees closely with a pKa value of 7.5 for Asp 26. The agreement between the experimental and theoretical changes in protein stability due to substitution of Asp 26 by alanine is also good. The local structure in the vicinity of Asp 26 in the low-pH crystal structure (with uncharged Asp 26) is hydrophobic, indicating that the aspartate would be highly destabilized. In theoretical calculations, the desolvation penalty for deprotonating Asp 26 in this environment is similar to the total protein folding energy. As a consequence, the Asp 26 pKa would be much greater than 7.5, and/or the protein might not fold. This suggests that a compensating process partially stabilizes the Asp 26 carboxyl group when it is charged. A simple model for this proposed, whereby the Lys 57 side chain rotates to form a salt bridge with Asp 26 when it is deprotonated.

Aspartic Acid↗
PubMed1991

Reconciling the magnitude of the microscopic and macroscopic hydrophobic effects.

The magnitude of the hydrophobic effect, as measured from the surface area dependence of the solubilities of hydrocarbons in water, is generally thought to be about 25 calories per mole per square angstrom (cal mol-1 A-2). However, the surface tension at a hydrocarbon-water interface, which is a "macroscopic" measure of the hydrophobic effect, is approximately 72 cal mol-1 A-2. In an attempt to reconcile these values, alkane solubility data have been reevaluated to account for solute-solvent size differences, leading to a revised "microscopic" hydrophobic effect of 47 cal mol-1 A-2. This value, when used in a simple geometric model for the curvature dependence of the hydrophobic effect, predicts a macroscopic alkane-water surface tension that is close to the macroscopic value.

Hydrocarbons↗
PubMed1991

Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons.

We demonstrate in this work that the surface tension, water-organic solvent, transfer-free energies and the thermodynamics of melting of linear alkanes provide fundamental insights into the nonpolar driving forces for protein folding and protein binding reactions. We first develop a model for the curvature dependence of the hydrophobic effect and find that the macroscopic concept of interfacial free energy is applicable at the molecular level. Application of a well-known relationship involving surface tension and adhesion energies reveals that dispersion forces play little or no net role in hydrophobic interactions; rather, the standard model of disruption of water structure (entropically driven at 25 degrees C) is correct. The hydrophobic interaction is found, in agreement with the classical picture, to provide a major driving force for protein folding. Analysis of the melting behavior of hydrocarbons reveals that close packing of the protein interior makes only a small free energy contribution to folding because the enthalpic gain resulting from increased dispersion interactions (relative to the liquid) is countered by the freezing of side chain motion. The identical effect should occur in association reactions, which may provide an enormous simplification in the evaluation of binding energies. Protein binding reactions, even between nearly planar or concave/convex interfaces, are found to have effective hydrophobicities considerably smaller than the prediction based on macroscopic surface tension. This is due to the formation of a concave collar region that usually accompanies complex formation. This effect may preclude the formation of complexes between convex surfaces.

Algorithms↗
PubMed1990

Electrical potential of transfer RNAs: codon-anticodon recognition.

Calculations of the electrostatic potentials were made around yeast elongator phenylalanine, aspartate tRNAs, and yeast initiator methionine tRNA in aqueous solution at physiological ionic strength. The calculations were carried out with a finite difference algorithm for solving the nonlinear Poisson-Boltzmann equation that incorporates the screening effects of the electrolyte, the exclusion of ions by the molecule, the molecular shape, and the different polarizabilities of the solvent and the tRNA. The initiator tRNA is surrounded by uniformly spaced contours of negative potential. The elongator tRNAs are also surrounded by a similar contour pattern except in the anticodon region where there is a pronounced "hole" in the potential surface. This hole is caused by an invagination of the potential contours, which also results in an increase in the local field strength. The effect of this hole is that the anticodon region in the elongator tRNAs is the least negative, or conversely the most positive, region of the molecule. This hole, which is not found when simple Coulombic potentials are used, is due both to the structure of the elongator tRNA anticodon loops and to the different polarizabilities of the solvent and tRNA. The existence of the potential hole in elongator tRNAs may account in part for their ability to associate with other negatively charged macromolecules, in particular mRNA. Moreover, it suggests that the anticodon loop of elongator tRNAs is the energetically most favorable point of approach of mRNA to tRNA.

Anticodon↗
PubMed1989

The electrostatic potential of B-DNA.

Electrostatic potentials around DNA are obtained by solving the nonlinear Poisson-Boltzmann (PB) equation. The detailed charge distribution of the DNA and the different polarizabilities of the macromolecule and solvent are included explicitly in the calculations. The PB equation is solved using extensions of a finite difference approach applied previously to proteins. Electrical potentials and ion concentrations are compared to those obtained with simpler models. It is found that the shape of the dielectric boundary between the macromolecule and solvent has significant effects on the calculated potentials near the surface, particularly in the grooves. Sequence-specific patterns are found, the most surprising result being the existence of positive regions of potential near the bases in both the major and minor grooves. The effect of solvent and ionic atmosphere screening of phosphate-phosphate repulsions is studied, and an effective dielectric function, appropriate for molecular mechanics simulations, is derived.

DNA↗
PubMed1986

Synthesis and application of a poly(ethylene glycol)-antibody affinity ligand for cell separations in aqueous polymer two-phase systems.

The possibility of producing biospecific affinity ligands for separating cells in two polymer aqueous phase systems on the basis of cell surface antigens was investigated. Rabbit anti-human erythrocyte IgG was reacted with cyanuric chloride-activated monomethyl poly(ethylene glycol) (PEG) fractions (molecular weights approximately 200, 1900, and 5000) at various molar ratios of PEG to protein lysine groups. The partition coefficient of the protein in a Dextran/PEG two-phase system increased with increasing degree of modification and increasing PEG molecular weight. There was a concomitant loss in ability to agglutinate human erythrocytes. The ability of the modified IgG to bind to a DEAE-cellulose column was almost eliminated by reaction with the PEG 5000, and was decreased to a lesser extent by PEG 1900. This PEG 1900-modified IgG substantially increased the partition of fresh or fixed human erythrocytes into the PEG-rich phase of a suitable phase system, while having no effect on rabbit cell partition. The partition increase could be inhibited by unmodified anti-human red cell IgG but not by nonspecific unmodified human IgG, demonstrating that the ligand effects were specific for the cell type against which the antibody was raised. A mixture of rabbit and human erythrocytes, which ordinarily have very similar partitions in the phase systems used, could be separated on a countercurrent distribution apparatus using the modified IgG. These results demonstrate the feasibility of producing immunologically specific affinity partition ligands for cell separation.

Affinity Labels↗
PubMed1983

Theory of the electrokinetic behavior of human erythrocytes.

We develop a theory of electrophoresis of human erythrocytes that predicts mobilities significantly smaller than those based on the classical Smoluchowski relation. In the classical treatment the charge is assumed to be spread uniformly on the hydrodynamic surface. The present model takes into account that most of the charge, due mainly to sialic acid, is contained in the glycocalyx. The glycocalyx is modeled as a permeable layer of polyelectrolyte molecules anchored to the cell membrane. The charge is assumed to be uniformly distributed throughout this layer. The fluid flow in the layer is treated as being dominated by Stokes friction arising from idealized polymer segments. The Navier-Stokes equations are solved to give the dependence of electroosomotic velocity with distance from the cell surface. An expression for the electrophoretic mobility is obtained which contains two parameters (a) the thickness of the glycocalyx and (b) the mean polymer segment radius. The best fit to experimental data is obtained if these are given the values 75 A and 7 A, respectively. Deviation from experimental data at low ionic strength (less than 0.05 M) occurs. However, this deviation is in the direction one would expect if at low ionic strength the polyelectrolyte layer expands slightly due to decreased charge shielding.

Anion Exchange Protein 1, Erythrocyte↗