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Results for “Solvent accessibility”

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PubMed1990

Stabilization of Z-DNA by demethylation of thymine bases: 1.3-A single-crystal structure of d(m5CGUAm5CG).

Methylation of cytosine bases at the C5 position has been known to stabilize Z-DNA. We had previously predicted from calculations of solvent-accessible surfaces that the methyl group at the same position of thymine has a destabilizing effect on Z-DNA. In the current studies, the sequence d(m5CGUAm5CG) has been crystallized and its structure solved as Z-DNA to 1.3-A resolution. A well-defined octahedral hexaaquomagnesium complex was observed to bridge the O4 oxygens of the adjacent uridine bases at the major groove surface, and four well-structured water molecules were found in the minor groove crevice at the d(UA) dinucleotide. These solvent interactions were not observed in the previously published Z-DNA structure of the analogous d(m5CGTAm5CG) sequence. A comparison of the thymine and uridine structures supports our prediction that demethylation of thymine bases helps to stabilize Z-DNA. A comparison of this d(UA)-containing Z-DNA structure with the analogous d(TA) structure shows that access of the O4 position is hindered by the C5 methyl of thymine due to steric and hydrophobic inhibition. In the absence of the methyl group, a magnesium-water complex binds to and slightly affects the structure of the Z-DNA major groove surface. This perturbation of the solvent structure at the major groove surface is translated into a much larger 1.41-A widening of the minor groove crevice, thereby allowing the specific binding of two water molecules at well-defined sites of each internal d(UA) base pair. Possible mechanisms by which modifications at the major groove surface of Z-DNA can affect the solvent properties of the minor groove crevice are discussed.

5-Methylcytosine↗

PubMed1995

Algorithms for evaluating the long-range accessibility of protein surfaces.

Algorithms are presented for characterizing the long-range accessibilities of protein surfaces. First, we describe an analytical method for determining the maximum contact radius for each atom in a structure. The problem is simplified greatly by geometric inversion in a sphere, a type of conformal mapping. Second, we introduce the concept of diffusion accessibility of a protein surface, which we evaluate either by random-walk simulations or by numerical solution of the equations of diffusion with the protein acting as an adsorber. These two measures of exposure are compared to each other as well as to the more common notion of solvent accessibility. These new procedures provide longer-range descriptions of surface geometry which may be useful in docking studies and other areas where surface comparison is required.

Algorithms↗

PubMed2002

Role of the magnesium cation on antihypertensive molecule-human serum albumin binding: affinity chromatography approach.

The role of the Mg2+ cation on antihypertensive molecule binding on human serum albumin (HSA) was studied by affinity chromatography. The thermodynamic data corresponding to this binding were determined for a wide range of Mg2+ concentrations (c). For the nifedipine molecule, an increase in the Mg2+ concentration produced a decrease in binding due to a decrease in the electrostatic interactions. For verapamil and diltiazem, which have the highest solvent accessible surface area, the solute binding on HSA was divided into two Mg2+ concentration regions. For a low c value below c(c) (approximately 1.6 mmol/l), the binding dependence with c was similar to that of nifedipine. For c above c(c) the hydrophobic effect created in the bulk solvent associated with a decrease in the van der Waals interactions between the solute molecule and the HSA implied a decrease in its binding. These results showed that for patients with hypertension, an Mg2+ supplementation during treatment with these antihypertensive molecules can increase the active pharmacological molecule concentration.

Antihypertensive Agents↗

PubMed2002

Origins of protein denatured state compactness and hydrophobic clustering in aqueous urea: inferences from nonpolar potentials of mean force.

Free energies of pairwise hydrophobic association are simulated in aqueous solutions of urea at concentrations ranging from 0-8 M. Consistent with the expectation that hydrophobic interactions are weakened by urea, the association of relatively large nonpolar solutes is destabilized by urea. However, the association of two small methane-sized nonpolar solutes in water has the opposite tendency of being slightly strengthened by the addition of urea. Such size effects and the dependence of urea-induced stability changes on the configuration of nonpolar solutes are not predicted by solvent accessible surface area approaches based on energetic parameters derived from bulk-phase solubilities of model compounds. Thus, to understand hydrophobic interactions in proteins, it is not sufficient to rely solely on transfer experiment data that effectively characterize a single nonpolar solute in an aqueous environment but not the solvent-mediated interactions among two or more nonpolar solutes. We find that the m-values for the rate of change of two-methane association free energy with respect to urea concentration is a dramatically nonmonotonic function of the spatial separation between the two methanes, with a distance-dependent profile similar to the corresponding two-methane heat capacity of association in pure water. Our results rationalize the persistence of residual hydrophobic contacts in some proteins at high urea concentrations and explain why the heat capacity signature (DeltaC(P)) of a compact denatured state can be similar to DeltaC(P) values calculated by assuming an open random-coil-like unfolded state.

Calorimetry↗

PubMed2004

Volumetric properties of the formation of double stranded DNA: a nearest-neighbor analysis.

The kinetics of the helix-coil transition have been studied by performing UV-monitored melting and reannealing curves of DNA and analyzing the resultant hysteresis between these curves. The analysis assumes a single-step bimolecular transition with duplex formation defined as the forward reaction. Volume parameters of the helix-coil transition were obtained by measuring the pressure dependence of the rate constants from 5-200 MPa. The data were interpreted in terms of several possible nearest-neighbor models, ranging from one to eleven parameters. Twenty-four oligonucleotide duplexes 22 base pairs in length were used to solve for individual nearest-neighbor activation volumes and transition volumes. Statistically, the most valid fit of the volumetric data was obtained with a six-parameter model in which the directionality of the dinucleotide steps is not considered, for example, 5'AG/CT is the same as 5'GA/TC. The resultant transition volumes at 48 degrees C ranged from -7.1 +/- 0.8 mL/mol (GC/CG) to +2.9 +/- 0.3 mL/mol (AA/TT). The success of the six-parameter model suggests that the relative size of the nearest-neighbor dinucleotides is the most important factor determining the magnitude of the volumetric parameters. The finding that the magnitude of the volumetric parameters correlates with the change in the solvent accessible surface area of the bases during the helix-coil transition corroborates this hypothesis.

Algorithms↗

PubMed2001

Origin of apparent fast and non-exponential kinetics of lysozyme folding measured in pulsed hydrogen exchange experiments.

Folding of lysozyme at pH 5.2 is a complex processes. After rapid collapse (<1 ms) kinetic partitioning into a slow and fast folding pathway occurs. The fast pathway leads directly to the native structure (N), whereas the slow pathway goes through a partially folded intermediate (I(1)) with native-like secondary structure in the alpha-domain. This mechanism is in agreement with data from a large number of spectroscopic probes, from changes in the radius of gyration and from measurements on the time-course of the populations of the different species. Results from pulsed hydrogen exchange experiments, in contrast, revealed that the secondary structure of I(1) and of N is formed significantly faster than changes in spectroscopic properties occur and showed large variations in the protection kinetics of individual amide sites. We investigated the molecular origin of the rapid amide protection by quantitatively simulating all kinetic processes during the pulse-labeling experiments. Absorbance and fluorescence-detected folding kinetics showed that the early events in lysozyme folding are accelerated under exchange conditions (pH 9.2) and that a change in folding mechanism occurs due to the transient population of an additional intermediate (I(2)). This leads to kinetic competition between exchange and folding during the exchange pulse and to incomplete labeling of amide sites with slow intrinsic exchange rates. As a result, apparently faster and non-exponential kinetics of amide protection are measured in the labeling experiments. Our results further suggest that collapsed lysozyme (C) and I(1) have five and ten-times reduced free exchange rates, respectively, due to limited solvent accessibility.

Amides↗

PubMed2006

A simple, yet highly accurate, QSAR model captures the complement inhibitory activity of compstatin.

Compstatin is a 13-residue cyclic peptide inhibitor of complement activation that was originally identified through phage-mediated presentation of a peptide library to C3b. Recent efforts to improve its activity have led to a rich dataset of complement analogs, with the most active analog being approximately 260 times more active than the parent compstatin. In the present work, a highly transparent quantitative structure-activity relationship model (Radj2=0.89) with four parameters is presented that captures important physico-chemical and geometrical properties of the analog molecules with regard to activity. The number of aromatic bonds and hydrophobicity of the fourth residue of compstatin correlated strongly with activity. Also important were the hydrophobic patch size near the disulfide bond and the solvent-accessible surface area occupied by nitrogen atoms of basic amino acid residues.

Complement Activation↗

PubMed2002

Molecular dynamics simulations of calcium-free calmodulin in solution.

A 4-ns molecular dynamics simulation of calcium-free calmodulin in solution has been performed, using Ewald summation to treat electrostatic interactions. Our simulation results were mostly consistent with solution experimental studies, including NMR, fluorescence and x-ray scattering. The secondary structures within the N- and C-terminal domains were conserved in the simulation, with trajectory structures similar to the NMR-derived model structure 1CFD. However, the relative orientations of the domains, for which there are no NMR restraints, differed in details between the simulation and the 1CFD model. The most interesting information provided by the simulations is that the dynamics of calcium-free calmodulin in solution is dominated by slow rigid body reorientations of the domains. The interdomain distance fluctuated between 29 and 39 A, and interdomain orientation angle, defined as the pseudo-dihedral formed by the four calcium binding sites, varied between -2 degrees and 108 degrees. Similarly, the domain linker region also exhibited significant fluctuations, with its length varying in the 34-45 A range and its bend angle in the 10-100 degrees range. The simulations are in accord with fluorescence results suggesting that calcium-free calmodulin is more compact and more flexible than the calcium activated form. Surprisingly, quite similar solvent accessibilities of the hydrophobic patches were seen in the calcium-free trajectory described in this work and previously generated calcium-loaded calmodulin simulations. Thus, our simulations suggest a reexamination of the standard model of the structural change of calmodulin upon calcium binding, involving exposure of the hydrophobic patches to solvent.

Biophysical Phenomena↗

PubMed1997

Hydrophobic patches on protein subunit interfaces: characteristics and prediction.

Hydrophobic patches, defined as clusters of neighboring apolar atoms deemed accessible on a given protein surface, have been investigated on protein subunit interfaces. The data were taken from known tertiary structures of multimeric protein complexes. Amino acid composition and preference, patch size distribution, and patch contact complementarity across associating subunits were examined and compared with hydrophobic patches found on the solvent-accessible surface of the multimeric complexes. The largest or second largest patch on the accessible surface of the entire subunit was involved in multimeric interfaces in 90% of the cases. These results should prove useful for subunit design and engineering as well as for prediction of subunit interface regions.

Amino Acids↗

PubMed1991

Distance-dependent dielectric constants and their application to double-helical DNA.

Detailed studies of structures of biological macromolecules, even in simplified models, involve many costly and time-consuming calculations. Any thorough methods require sampling of an extremely large conformation and momentum space. Calculations of electrostatic interactions, which depend on many physical factors, such as the details of solvent, solvent accessibility in macromolecules, and molecular polarizability, are always developed in a compromise between more rigorous, detailed models and the need for immediate application to complicated biological systems. In this paper, a middle ground is taken between the more exact theoretical models and the simplest constant values for the dielectric constant. The effects of solvent, counterions, and molecular polarizability are incorporated through a set of adjustable parameters that should be determined from experimental conditions. Several previous forms for the dielectric function are compared with the new ones. The present methods use Langevin functions to span the region of dielectric constant between bulk solvent and cavity values. Application of such dielectric models to double-helical DNA is important because base-stacking preferences were previously demonstrated [A. Sarai, J. Mazur, R. Nussinov, and R. L. Jernigan (1988) Biochemistry, vol. 27, pp. 8498-8502] to be sensitive to the electrostatic formulation. Here we find that poly(dG).poly(dC) can be A form for high screening and B form for low screening. By contrast, poly(dA).poly(dT) can only take the B form. Base stacking is more sensitive to the form of the dielectric function than are the sugar-phosphate backbone conformations. Also in B form, the backbone conformations are not so affected by the base types as in A form.

Base Sequence↗

PubMed2006

Site-directed alkylation and the alternating access model for LacY.

In a functional lactose permease mutant from Escherichia coli (LacY) devoid of native Cys residues, almost every residue was replaced individually with Cys and tested for reactivity with the permeant alkylating agent N-ethylmaleimide in right-side-out membrane vesicles. Here we present the results in the context of the crystal structure of LacY. Engineered Cys replacements located near or within the inward-facing hydrophilic cavity or at other solvent-accessible positions in LacY react well with this alkylating agent. Cys residues facing the low dielectric of the membrane or located in tightly packed regions of the structure react poorly. Remarkably, in the presence of ligand, increased reactivity is observed with Cys replacements located predominantly on the periplasmic side of the sugar-binding site. In contrast, other Cys replacements largely on the cytoplasmic side of the binding site exhibit decreased reactivity. Furthermore, both sets of Cys replacements in the putative cavities are located at the periplasmic (increased reactivity) and cytoplasmic (decreased reactivity) ends of the same helices and distributed in a pseudosymmetrical manner. The results are consistent with a model in which the single sugar-binding site in the approximate middle of the molecule is alternately exposed to either side of the membrane due to opening and closing of cytoplasmic and periplasmic hydrophilic cavities.

Alkylating Agents↗

PubMed1998

Solvent isotope effect on thermodynamics of hydration.

Partial molar heat capacities of five linear alcohols (methanol, ethanol, n-propanol, n-butanol, n-pentanol) and five N-substituted amides (n-propionamide, N-methylformamide, N-methylacetamide, N-methylpropionamide, N-ethylacetamide) in aqueous D(2)O solution have been measured at 25 degrees C. The heat capacities of transfer of these compounds from H(2)O to D(2)O were calculated using previously reported (Makhatadze et al., Biophys. Chem. 64 (1997) 93) values of partial heat capacities of alcohols and amides in aqueous H(2)O solutions. It is shown that the sign and magnitude of the heat capacity change upon transfer from H(2)O to D(2)O depends on the relative amount of polar and non-polar solvent accessible surface areas of solute. Analysis shows that transfer of non-polar surface from H(2)O to D(2)O is accompanied by a positive heat capacity change. In contrast, transfer of polar surface from H(2)O to D(2)O occurs with negative heat capacity change. Estimates show that the solvent isotope effect on the heat capacity changes upon protein unfolding can be predicted using the changes of the polar and non-polar surface area changes upon protein unfolding and the transfer data of model compounds. Analysis of the thermodynamic functions of transfer of non-polar compounds from H(2)O to D(2)O shows puzzling behavior which contradicts current definitions of the hydrophobic effect.

Journal Article↗

PubMed1995

Three-dimensional structure of the human 'protective protein': structure of the precursor form suggests a complex activation mechanism.

BACKGROUND: The human 'protective protein' (HPP) forms a multi-enzyme complex with beta-galactosidase and neuraminidase in the lysosomes, protecting these two glycosidases from degradation. In humans, deficiency of HPP leads to the lysosomal storage disease galactosialidosis. Proteolytic cleavage of the precursor form of HPP involves removal of a 2 kDa excision peptide and results in a carboxypeptidase activity. The physiological relevance of this activity is, as yet, unknown. RESULTS: The crystal structure of the 108 kDa dimer of the precursor HPP has been elucidated by making extensive use of twofold density averaging. The monomer consists of a 'core' domain and a 'cap' domain. Comparison with the distantly related wheat serine carboxypeptidase dimer shows that the two subunits in the HPP dimer differ by 15 degrees in mutual orientation. Also, the helical subdomain forming part of the cap domains is very different. In addition, the HPP precursor cap domain contains a 'maturation' subdomain of 49 residues which fills the active-site cleft. Merely removing the 'excision' peptide located in the maturation subdomain does not render the catalytic triad solvent accessible. CONCLUSIONS: The activation mechanism of HPP is unique among proteases with known structure. It differs from the serine proteases in that the active site is performed in the zymogen, but is blocked by a maturation subdomain. In contrast to the zinc metalloproteases and aspartic proteases, the chain segment physically rendering the catalytic triad solvent inaccessible in HPP is not cleaved off to form the active enzyme. The activation must be a multi-step process involving removal of the excision peptide and major conformational changes of the maturation subdomain, whereas the conformation of the enzymatic machinery is probably almost, or completely, unaffected.

Amino Acid Sequence↗

PubMed2002

A novel approach for identifying the surface atoms of macromolecules.

A significant number of atoms lie buried beneath the "molecular surface" of proteins and other biologic macromolecules. Interactions between ligands and these macromolecules are dominated by interactions with the "surface atoms". Although interactions with the "buried" or interior atoms of the macromolecule certainly contribute to the total intermolecular interaction energy, many computer-assisted drug design (CADD) strategies can benefit from the identification of those atoms "on the surface" of proteins and other macromolecules. We have developed a simple, yet novel method to distinguish the surface atoms of macromolecules from the interior atoms which is based on computing the atomic contributions to the solvent-accessible surface (SAS) area. This report describes that method and demonstrates that it compares very favorably with four alternative methods.

Computer Graphics↗

PubMed2004

Two different proteins that compete for binding to thrombin have opposite kinetic and thermodynamic profiles.

Thrombin binds thrombomodulin (TM) at anion binding exosite 1, an allosteric site far from the thrombin active site. A monoclonal antibody (mAb) has been isolated that competes with TM for binding to thrombin. Complete binding kinetic and thermodynamic profiles for these two protein-protein interactions have been generated. Binding kinetics were measured by Biacore. Although both interactions have similar K(D)s, TM binding is rapid and reversible while binding of the mAb is slow and nearly irreversible. The enthalpic contribution to the DeltaG(bind) was measured by isothermal titration calorimetry and van't Hoff analysis. The contribution to the DeltaG(bind) from electrostatic steering was assessed from the dependence of the k(a) on ionic strength. Release of solvent H(2)O molecules from the interface was assessed by monitoring the decrease in amide solvent accessibility at the interface upon protein-protein binding. The mAb binding is enthalpy driven and has a slow k(d). TM binding appears to be entropy driven and has a fast k(a). The favorable entropy of the thrombin-TM interaction seems to be derived from electrostatic steering and a contribution from solvent release. The two interactions have remarkably different thermodynamic driving forces for competing reactions. The possibility that optimization of binding kinetics for a particular function may be reflected in different thermodynamic driving forces is discussed.

Allosteric Regulation↗

PubMed2005

Multiple subsets of side-chain packing in partially folded states of alpha-lactalbumins.

Photochemically induced dynamic nuclear polarization NMR pulse-labeling techniques have been used to obtain detailed information about side-chain surface accessibilities in the partially folded (molten globule) states of bovine and human alpha-lactalbumin prepared under a variety of well defined conditions. Pulse labeling involves generating nuclear polarization in the partially folded state, rapidly refolding the protein within the NMR sample tube, then detecting the polarization in the well dispersed native-state spectrum. Differences in the solvent accessibility of specific side chains in the various molten globule states indicate that the hydrophobic clusters involved in stabilizing the alpha-lactalbumin fold can be formed from interactions between a variety of different hydrophobic residues in both native and non-native environments. The multiple subsets of hydrophobic clusters are likely to result from the existence of distinct but closely related local minima on the free-energy landscape of the protein and show that the fold and topology of a given protein may be formed from degenerate groups of side chains.

Amino Acid Sequence↗

PubMed1996

Serpin alpha 1proteinase inhibitor probed by intrinsic tryptophan fluorescence spectroscopy.

Various conformational forms of the archetypal serpin human alpha 1proteinase inhibitor (alpha 1PI), including ordered polymers, active and inactive monomers, and heterogeneous aggregates, have been produced by refolding from mild denaturing conditions. These forms presumably originate by different folding pathways during renaturation, under the influence of the A and C sheets of the molecule. Because alpha 1PI contains only two Trp residues, at positions 194 and 238, it is amenable to fluorescence quenching resolved spectra and red-edge excitation measurements of the Trp environment. Thus, it is possible to define the conformation of the various forms based on the observed fluorescent properties of each of the Trp residues measured under a range of conditions. We show that denaturation in GuHCl, or thermal denaturation in Tris, followed by renaturation, leads to the formation of polymers that contain solvent-exposed Trp 238, which we interpret as ordered head-to-tail polymers (A-sheet polymers). However, thermal denaturation in citrate leads to shorter polymers where some of the Trp 238 residues are not solvent accessible, which we interpret as polymers capped by head-to-head interactions via the C sheet. The latter treatment also generates monomers thought to represent a latent form, but in which the environment of Trp 238 is occluded by ionized groups. These data indicate that the folding pathway of alpha 1PI, and presumably other serpins, is sensitive to solvent composition that affects the affinity of the reactive site loop for the A sheet or the C sheet.

Humans↗

PubMed2000

Biotinylation of substituted cysteines in the nicotinic acetylcholine receptor reveals distinct binding modes for alpha-bungarotoxin and erabutoxin a.

Although previous results indicate that alpha-subunit residues Trp(187), Val(188), Phe(189), Tyr(190), and Pro(194) of the mouse nicotinic acetylcholine receptor are solvent-accessible and are in a position to contribute to the alpha-bungarotoxin (alpha-Bgtx) binding site (Spura, A., Russin, T. S., Freedman, N. D., Grant, M., McLaughlin, J. T., and Hawrot, E. (1999) Biochemistry 38, 4912-4921), little is known about the accessibility of other residues within this region. By determining second-order rate constants for the reaction of cysteine mutants at alpha184-alpha197 with the thiol-specific biotin derivative (+)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctanediamine , we now show that only very subtle differences in reactivity (approximately 10-fold) are detectable, arguing that the entire region is solvent-exposed. Importantly, biotinylation in the presence of saturating concentrations of the long neurotoxin alpha-Bgtx is significantly retarded for positions alphaW187C, alphaF189C, and reduced wild-type receptors (alphaCys(192) and alphaCys(193)), further emphasizing their major contribution to the alpha-Bgtx binding site. Interestingly, although biotinylation of position alphaV188C is not affected by the presence of alpha-Bgtx, erabutoxin a, which is a member of the short neurotoxin family, inhibits biotinylation at position alphaV188C, but not at alphaW187C or alphaF189C. Taken together, these results indicate that short and long neurotoxins establish interactions with distinct amino acids on the nicotinic acetylcholine receptor.

Amino Acid Substitution↗