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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

The influence of phospholipid polar groups on gramicidin channels

The influence of well-defined changes in the polar part of phospholipid molecules on the properties of black lipid membranes was studied using a series of phospholipids with identical hydrocarbon chains, but systematically changed polar groups. The hydrocarbon tails of the lipids under study were composed of 1,2-dipentadecylmethylidene glycerol. The polar parts differed in the degree of $\text{N-}\text{methylation}$ and comprised phosphocholine, $\text{-N,N-}\text{dimethylethanolamine}$, $\text{-N-}\text{methylethanolamine}$ and ethanolamine. Stable black lipid membranes could be formed with the solvents octane, decane, dodecane, tetradecane and hexadecane. The properties of gramicidin-induced single ionic channels changed systematically in membranes from the phosphatidylcholine to the phosphatidylethanolamine analogue, as indicated by an increase in the amplitude A of the unit conductance step and a decrease in the average channel life-time or duration τ. The series of τ-values was opposite to that expected from hydrocarbon thickness (specific capacitance). It is suggested that the surface tension γ is a relevant parameter for the prediction of τ-values.     

Detecting groups of coevolving positions in a molecule: a clustering approach

Background

Although the patterns of co-substitutions in RNA is now well characterized, detection of coevolving positions in proteins remains a difficult task. It has been recognized that the signal is typically weak, due to the fact that (i) amino-acid are characterized by various biochemical properties, so that distinct amino acids changes are not functionally equivalent, and (ii) a given mutation can be compensated by more than one mutation, at more than one position.

Results

We present a new method based on phylogenetic substitution mapping. The two above-mentioned problems are addressed by (i) the introduction of a weighted mapping, which accounts for the biochemical effects (volume, polarity, charge) of amino-acid changes, (ii) the use of a clustering approach to detect groups of coevolving sites of virtually any size, and (iii) the distinction between biochemical compensation and other coevolutionary mechanisms. We apply this methodology to a previously studied data set of bacterial ribosomal RNA, and to three protein data sets (myoglobin of vertebrates, S-locus Receptor Kinase and Methionine Amino-Peptidase).

Conclusion

We succeed in detecting groups of sites which significantly depart the null hypothesis of independence. Group sizes range from pairs to groups of size ? 10, depending on the substitution weights used. The structural and functional relevance of these groups of sites are assessed, and the various evolutionary processes potentially generating correlated substitution patterns are discussed.     

Charge environments around phosphorylation sites in proteins

Background  

Phosphorylation is a central feature in many biological processes. Structural analyses have identified the importance of charge-charge interactions, for example mediating phosphorylation-driven allosteric change and protein binding to phosphopeptides. Here, we examine computationally the prevalence of charge stabilisation around phosphorylated sites in the structural database, through comparison with locations that are not phosphorylated in the same structures.     

Pregnanolone isomers,pregnenolone and their polar conjugates around parturition

The levels of four pregnanolone isomers and their polar conjugates and pregnenolone sulfate were measured in the plasma of 13 and 7 women at delivery with subarachnoidal and epidural analgesia, respectively, and in corresponding samples of umbilical plasma using a simple quadrupole GC/MS system with electron impact ionization (pregnenolone isomers), RIA following HPLC separation (pregnenolone) and specific RIA (pregnanolone sulfate). The concentration of epipregnanolone (3beta-hydroxy-5beta-pregnan-20-one) in both maternal and umbilical plasma was much lower than that of other pregnanolone isomers. The levels of 3beta-hydroxy-pregnanolone isomers were significantly higher in the umbilical plasma than in the maternal, while the differences in 3alpha-hydroxy-isomers were insignificant. The differences in conjugates were insignificant with the exception of allopregnanolone, the levels of which were lower in umbilical plasma. In all the pregnanolone isomers, a significantly lower conjugated/unconjugated steroid ratio was found in the umbilical plasma than in the maternal plasma. In addition, time profiles of the steroids were measured around parturition and in the postpartum period in the maternal serum. Similarly, the levels of polar conjugates of all pregnanolone isomers were followed during parturition. Changes in concentrations of free steroids exhibited a similar pattern, with a fall primarily within the first hour after delivery. The decrease in conjugated steroids was shifted to the interval within the first hour and first day after delivery, and the changes were more pronounced. The time profiles of the conjugated/free steroid ratio exhibited a significant decrease within the first hour and the first day after delivery in all of the isomers investigated. A decrease was also observed in the ratio of 3alpha/3beta-isomers and 5alpha/5beta-isomers around parturition. The possible physiological consequences of the findings are indicated.     

A molecular dynamics study of solvent behavior around a protein

The solvent structure and behavior around a protein were examined by analyzing a trajectory of molecular dynamics simulation of thetrp-holorepressor in a periodic box of water. The calculated selfdiffusion coefficient indicated that the solvent within 10 Å of the protein had lower mobility. Examination of the solvent diffusion around different atoms of different kinds of residues showed no general tendency. Thisfact suggested that the solvent mobility is not influenced significantly bythe kind of the atom or residue they solvated. Distribution analysis aroundthe protein revealed two peaks of water oxygen: a sharp one at 2.8 Å around polar and charged atoms and a broad one at ～3.4 Å aroundapolar atoms. The former was stabilized by water–protein hydrogen bonds, and the latter was stabilized by water-lwater hydrogen bonds, suggesting the existence of a hydrophobic shell. An analysis of protein atom–water radial distribution functions confirmed these shell structures around polar or charged atoms and apolar ones. © 1993 Wiley-Liss, Inc.     

Atom-wise statistics and prediction of solvent accessibility in proteins

In this work, we explore a novel method to broaden the scope of sequence-based predictions of solvent accessibility or accessible surface area (ASA) to the atomic level. All 167 heavy atoms from the 20 types of amino acid residues in proteins have been studied. An analysis of ASA distribution of these atomic groups in different proteins has been performed and rotamer-style libraries have been developed. We observe that the ASA of some atomic groups (e.g., backbone C and N atoms) can be estimated from the sequence environment within a mean absolute error of 2-3 angstroms(2). However, some side chain atoms such as CG in Pro, NH1 in Arg and NE2 in Gln show a strong variability making it more difficult to estimate their ASA from sequence environment. In general, the prediction of ASA becomes more difficult for atomic positions at the side chain extremities of long amino acid residues (aromatic side chain terminals being the exception). Several atomic groups are frequently exposed to solvent. Some of them have a bimodal distribution, suggesting two stable conformations in terms of their solvent exposure. More detailed understanding and prediction of solvent accessibility, i.e., at an atomic level is expected to help in bioinformatics approaches to structure prediction, functional relevance of atomic solvent accessibilities and other interaction analyses.