Local effects in hydrophobicity. Electrostatic interactions in the restructuring of highly polar solvent around less polar solute

Two simplifying assumptions are frequently used in the biophysical chemistry of aqueous solutions: (i) a dielectric mediates the interactions of polar and ionic molecules in aqueous phases and (ii) the dielectric constant of this medium is high and uniform up to molecular surfaces. Because of their great utility in rationalizing simple electrostatic and dielectric effects in such polar systems, it is important to examine whether these assumptions also lead to deductions that are locally consistent with the solvent restructuring observed in hydrophobic phenomena. In this paper, using a model polar fluid system, these macroscopic assumptions are applied to the rigorous, microscopic nonlinear integral equation for Wki, the potential of mean force between two adjacent polar molecules. In systems of high dielectric constant, linearization of Boltzmann exponentials and approximation of three-molecule potentials of mean force by superposition of two-molecule potentials permit reduction to a linear integral equation for Wki. It is shown that the strictly local electrostatic contributions to Wki exert an effect that is qualitatively similar to the global screening effect of a dielectric medium. Through the relation between Wki and configurational probabilities, it is further found that reducing the polarity of a molecule in a polar fluid shifts local pair probability density from energetically unfavorable to energetically favorable two-molecule configurations. This general effect, which clearly promotes local structure, would augment more specific hydrophobic mechanisms in aqueous systems. Thus, the assumptions upon which the highly successful Debye-Hückel and Onsager models are supported lead also to deductions about local structure that are consistent with hydrophobic structure enhancement.     

Heat capacity changes upon burial of polar and nonpolar groups in proteins

In this paper we address the question of whether the burial of polar and nonpolar groups in the protein locale is indeed accompanied by the heat capacity changes, DeltaC(p), that have an opposite sign, negative for nonpolar groups and positive for polar groups. To accomplish this, we introduced amino acid substitutions at four fully buried positions of the ubiquitin molecule (Val5, Val17, Leu67, and Gln41). We substituted Val at positions 5 and 17 and Leu at position 67 with a polar residue, Asn. As a control, Ala was introduced at the same three positions. We also replaced the buried polar Gln41 with Val and Leu, nonpolar residues that have similar size and shape as Gln. As a control, Asn was introduced at Gln41 as well. The effects of these amino acid substitutions on the stability, and in particular, on the heat capacity change upon unfolding were measured using differential scanning calorimetry. The effect of the amino acid substitutions on the structure was also evaluated by comparing the (1)H-(15)N HSQC spectra of the ubiquitin variants. It was found that the Ala substitutions did not have a considerable effect on the heat capacity change upon unfolding. However, the substitutions of aliphatic side chains (Val or Leu) with a polar residue (Asn) lead to a significant (> 30%) decrease in the heat capacity change upon unfolding. The decrease in heat capacity changes does not appear to be the result of significant structural perturbations as seen from the HSQC spectra of the variants. The substitution of a buried polar residue (Gln41) to a nonpolar residue (Leu or Val) leads to a significant (> 25%) increase in heat capacity change upon unfolding. These results indicate that indeed the heat capacity change of burial of polar and nonpolar groups has an opposite sign. However, the observed changes in DeltaC(p) are several times larger than those predicted, based on the changes in water accessible surface area upon substitution.     

Influence of solvent and configuration of residues at positions 2 and 3 on distance and mobility of pharmacophore groups at positions 1 and 4 in cyclic enkephalin analogues

The analgesic activity of opioid peptides is mainly connected with their affinity and selectivity for the mu-receptors. The biological activity of cyclic opioid analogues depends on mutual orientation and conformational freedom of aromatic pharmacophore groups at positions 1 and 4. The distance and distance distributions between chromophores at positions 1 [Phe(p-NO(2)), p-nitrophenylalanine] and 4 [Nal, beta-(2-naphthyl)alanine], which constitute an energy donor-acceptor pair, were calculated based on measured fluorescence intensity decays of a donor (Nal). The influence of the solvent and configuration of the residues at position 2 and 3 on donor-acceptor distance distribution and mobility of pharmacophore groups at position 1 and 4 in cyclic enkephalin analogues are discussed.     

Crystallographic study of hydration of an internal cavity in engineered proteins with buried polar or ionizable groups

Although internal water molecules are essential for the structure and function of many proteins, the structural and physical factors that govern internal hydration are poorly understood. We have examined the molecular determinants of internal hydration systematically, by solving the crystal structures of variants of staphylococcal nuclease with Gln-66, Asn-66, and Tyr-66 at cryo (100 K) and room (298 K) temperatures, and comparing them with existing cryo and room temperature structures of variants with Glu-66, Asp-66, Lys-66, Glu-92 or Lys-92 obtained under conditions of pH where the internal ionizable groups are in the neutral state. At cryogenic temperatures the polar moieties of all these internal side chains are hydrated except in the cases of Lys-66 and Lys-92. At room temperature the internal water molecules were observed only in variants with Glu-66 and Tyr-66; water molecules in the other variants are probably present but they are disordered and therefore undetectable crystallographically. Each internal water molecule establishes between 3 and 5 hydrogen bonds with the protein or with other internal water molecules. The strength of interactions between internal polar side chains and water molecules seems to decrease from carboxylic acids to amides to amines. Low temperature, low cavity volume, and the presence of oxygen atoms in the cavity increase the positional stability of internal water molecules. This set of structures and the physical insight they contribute into internal hydration will be useful for the development and benchmarking of computational methods for artificial hydration of pockets, cavities, and active sites in proteins.     

Modelling of proteins in membranes

This review describes some recent theories and simulations of mesoscopic and microscopic models of lipid membranes with embedded or attached proteins. We summarize results supporting our understanding of phenomena for which the activities of proteins in membranes are expected to be significantly affected by the lipid environment. Theoretical predictions are pointed out, and compared to experimental findings, if available. Among others, the following phenomena are discussed: interactions of interfacially adsorbed peptides, pore-forming amphipathic peptides, adsorption of charged proteins onto oppositely charged lipid membranes, lipid-induced tilting of proteins embedded in lipid bilayers, protein-induced bilayer deformations, protein insertion and assembly, and lipid-controlled functioning of membrane proteins.     

Site fidelity of Svalbard polar bears revealed by mark-recapture positions

Quantifying the degree of site fidelity in polar bears’ (Ursus maritimus) use of denning and mating areas in spring is of considerable interest for both basic and applied purposes. We analyzed 276 spring-to-spring movements (displacements) of 178 polar bears obtained from mark-recapture in the period 1987–2010 in Svalbard, Norway. Male and female subadults and adults showed site fidelity (only subadult females non-significantly) when their movements were compared to a scenario of random movements between all capture locations. The median observed displacement was 47.6 km (bootstrapped 95 % CI: 38.6–57.8 km), significantly smaller than the median potential displacement for random movements of 206.3 km (bootstrapped 95 % CI: 187.3–219.6 km). Subadult females tended to have the longest displacements, followed by adult males and adult females. However, large individual variation both in displacement distances and recapture frequency tended to blur age and sex differences. Analysis restricted to one movement for each bear showed that the difference between adult males and adult females was small and non-significant. This indicates that findings based on telemetry, which is almost always restricted to females, may be relatively representative of the whole adult Barents Sea population in the spring season.     

Preferential and absolute interactions of solvent components with proteins in mixed solvent systems

An equation is derived relating total and preferential interactions of solvent components with macromolecules in a three-component system. Application of this equation to literature data shows that binding of non-polar solvents to proteins parallels the unfolding of the latter. This observation is discussed in terms of local inter-residue and residue-solvent interactions.     

Organization of polar groups of 9 kd calbindin around Ca2+ ions bound to the protein: a microdielectric study

Using a simple model of linear response of polarizable centers to an electric field, function delta is defined to characterize structural organization of protein polar groups. The function makes it possible to detect specific structures of nonuniformly distributed and mutually coupled groups that can transmit local structural and charge density perturbations, induced by an ion over long distances to functionally active sites of a protein molecule. In 9 kd calbinding (a small protein from the troponin C superfamily) two structural chains have been demonstrated that link together both Ca2+ ions coordinated by the protein. The chains form a rigid structure stabilized by coordination of one of the ions, so that binding of the other is promoted. Such a structure is probably common to all members of the superfamily and plays an important role in the mechanism of calcium binding by these proteins.     

Fixation of deleterious mutations at critical positions in human proteins

Deleterious mutations associated with human diseases are predominantly found in conserved positions and positions that are essential for the structure and/or function of proteins. However, these mutations are purged from the human population over time and prevented from being fixed. Contrary to this belief, here I show that high proportions of deleterious amino acid changing mutations are fixed at positions critical for the structure and/or function of proteins. Similarly, a high rate of fixation of deleterious mutations was observed in slow-evolving amino acid positions of human proteins. The fraction of deleterious substitutions was found to be two times higher in relatively conserved amino acid positions than in highly variable positions. This study also found fixation of a much higher proportion of radical amino acid changes in primates compared with rodents and artiodactyls in slow-evolving positions. Previous studies observed a higher proportion of nonsynonymous substitutions in humans compared with other mammals, which was taken as indirect evidence for the fixation of deleterious mutations in humans. However, the results of this investigation provide direct evidence for this prediction by suggesting that the excess nonsynonymous mutations fixed in humans are indeed deleterious in nature. Furthermore, these results suggest that studies on disease-associated mutations should consider that a significant fraction of such deleterious mutations has already been fixed in the human genome, and thus, the effects of new mutations at those amino acid positions may not necessarily be deleterious and might even result in reversion to benign phenotypes.     

Arrangement of disulfide bridges and positions of sulfhydryl groups in tetanus toxin

Tetanus toxin is a 151-kDa protein. The complete amino acid sequence is known. The mature toxin is made up of two peptide chains and contains 10 half-cystine residues. Treatment with 4-vinylpyridine in the presence of 6 M guanidine converted six of them into S-pyridylethyl cysteine residues as determined by amino acid analysis. When alkylation was preceded by mercaptolysis, all 10 half-cystine residues were recovered in the S-pyridylethylated form. It was therefore concluded that the toxin contains six sulfhydryl groups and two disulfide bonds. The positions of the residues carrying sulfhydryl groups and of those involved in disulfide bridges were determined by labelling of the toxin alternatively with 4-vinylpyridine or with 4-dimethylaminoazobenzene-4'-iodoacetamide (DABIA), directly or after mercaptolysis. The toxin derivatives were cleaved with cyanogen bromide and the elution patterns in reversed-phase HPLC compared. The chromatography components were identified by N-terminal amino acid sequence and amino acid composition. In the chromatography of the non-mercaptolysed, DABIA-treated sample four chromophore-carrying components were detected which could be demonstrated by N-terminal sequence analysis to correspond to six half-cystine-containing cyanogen bromide fragments. In the mercaptolysed, DABIA-treated sample three additional chromophore-carrying components were present, corresponding to two previously disulfide-linked cyanogen bromide fragments and one fragment which had contained an internal disulfide bridge. The HPLC patterns showed characteristic differences as the DABIA-labelled fragments were considerably more hydrophobic than the corresponding vinylpyridine-labelled fragments. It was established that the half-cystine residues in positions 26, 185, 198, 311, 868, and 1300 are present in the sulfhydryl form, that those in positions 438 and 466 are disulfide-bridged, thereby connecting the light and heavy chains of the toxin, and that those in positions 1076 and 1092 are disulfide-bridged, thereby giving rise to a loop in the heavy chain. During the progress of the investigations about 20% of the amino acid sequence previously predicted from DNA analysis was confirmed by protein-chemical methods.     

Local dielectric properties around polar region of lipid bilayer membranes

Summary Local dielectric constant was evaluated from the Stokes shifts of fluorescence spectra ofl--dansylphosphatidylethanolamine (DPE) incorporated into liposomes made of synthetic phosphatidylcholine (dipalmitoyl or distearoyl) or bovine brain phosphatidylserine. The evaluation was established as follows. First, the Stokes shift of DPE was assured to follow Mataga-Lippert's equation and was a function of the dielectric constant and the refractive index in some standard organic solvents. Second, the change of the refractive index did not contribute much to the change in the Stokes shift. Third, the time resolved fluorescence depolarization of DPE in liposomes showed that the cone wobbling diffusion was rapid relative to the fluorescence lifetime and therefore that the dielectric relaxation did not affect the evaluation of the constant in the polar region of membranes. We then investigated the characteristics of the local dielectric constant in the polar region of the lipid bilayer and found that the dielectric constant varies between 4 and 34 depending upon calcium binding and also gel/liquid-crystal phase transition. Such large changes of the local dielectric constant were further correlated with the dynamic structure of lipid bilayer membranes measured by conventional fluorescence depolarization techniques. The large changes of dielectric constant around the polar region suggest that electrostatic interactions at this region can be altered 10-fold by such ionic or thermotropic factors and therefore that local dielectric properties can play crucial roles in membrane functions.