The membrane as an environment of minimal interconversion. A circular dichroism study on the solvent dependence of the conformational behavior of gramicidin in diacylphosphatidylcholine model membranes

The conformation of gramicidin in diacylphosphatidylcholine model membranes was investigated as a function of the solvent in which peptide and lipid are initially codissolved. By use of circular dichroism it is demonstrated that, upon removal of the solvent and hydration of the mixed gramicidin/lipid film, it is the conformational behavior of the peptide in the organic solvent that determines its final conformation in dimyristoylphosphatidylcholine model membranes. As a consequence, parameters that influence the conformation of the peptide in the solvent also play an essential role, such as the gramicidin concentration and the rate of interconversion between different conformations. Of the various solvents investigated, only with trifluoroethanol is it possible directly to incorporate gramicidin entirely in the beta 6.3-helical (channel) configuration. It is also shown that the conformation of gramicidin in the membrane varies with the peptide/lipid ratio, most likely as a result of intermolecular gramicidin-gramicidin interactions at higher peptide/lipid ratios, and that heat incubation leads to a conformational change in the direction of the beta 6.3-helical conformation. Using lipids with an acyl chain length varying from 12 carbon atoms in dilauroylphosphatidylcholine to 22 carbon atoms in dierucoylphosphatidylcholine, it was possible to investigate the acyl chain length dependence of the gramicidin conformation in model membranes prepared from these lipids with the use of different solvent systems. It is demonstrated for each solvent system that the distribution between different conformations is relatively independent of the acyl chain length but that the rate at which the conformation converts toward the beta 6.3-helical configuration upon heating of the samples is affected by the length of the acyl chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural asymmetry in the CTP-liganded form of aspartate carbamoyltransferase from Escherichia coli

The protein and solvent structure of the CTP-liganded form of aspartate carbamoyltransferase from Escherichia coli yields an R-factor of 0.155 for data to a resolution of 2.6 A. The model has 7353 protein atoms, 945 sites for solvent, and two molecules of CTP. A total of 25 of the 912 residues of the model exist in more than one conformation. The root-mean-square deviation of bond lengths and angles from their ideal values is 0.013 A and 2.1 degrees, respectively. The model reported here reflects a correction in the trace of the regulatory chain. One molecule of CTP binds to each of the two regulatory chains of the asymmetric unit of the crystal. The interactions between the pyrimidine of each CTP molecule and the protein are similar. The 4-amino group of CTP binds to the carbonyl groups of residues 89 (tyrosine) and 12 (isoleucine) of the regulatory chain. The nitrogen of position 3 of the pyrimidine binds to the amide group of residue 12; the 2-keto group binds to lysine 60. The 2'-OH group of the ribose forms hydrogen bonds with lysine 60 and the carbonyl group of residue 9 (valine). The binding of the phosphate groups of CTP to the regulatory chain probably reflects an incomplete association of CTP with the enzyme at pH 5.8. A lattice contact influences the interaction between the triphosphate group of one CTP molecule and the protein. For the other CTP molecule, only lysine 94 binds to the phosphate groups of CTP. Of the two regulatory and two catalytic chains of the asymmetric unit of the crystal, there are only two significant violations of non-crystallographic symmetry. The active site in the vicinity of arginine 54 of one catalytic chain is larger than the active site of its non-crystallographic mate. The "expanded" cavity accommodates four solvent molecules in the vicinity of arginine 54 as opposed to two molecules of water for the "contracted" cavity. Furthermore, arginine 54 in the "expanded" pocket adopts two conformations, either hydrogen-bonding to glutamate 86 or to the phenolic oxygen atom of tyrosine 98; residues 86 and 98 are in a catalytic chain related by 3-fold symmetry to the catalytic chain of arginine 54. In the "contracted" pocket, arginine 54 binds only to glutamate 86.(ABSTRACT TRUNCATED AT 400 WORDS)     

Amylose optical rotation in some mixed solvent systems

This paper extends a previous study in which a discontinuity in the specific rotation of open chain α-l,4-linked glucopyranosides in the water–dimethyl sulfoxide (H₂O–DMSO) system was attributed to a symmetry change about a polymer chain segment. Optical rotation of amylose, cyclohexamylose, methyl β-maltoside, and dextran was measured in the following mixed solvent systems: formamide–dimethyl sulfoxide (F-DMSO), ethylenediamine–dimethyl sulfoxide (E–DMSO), and hexamethylphosphoramide–dimethyl sulfoxide (HMPA–DMSO). Refractive index measurements were used in an attempt to detect hydrogen bonding between solvent components. The specific rotation of amylose corrected for variation in refractive index (CSR), as a function of solvent composition, showed a discontinuity at solvent compositions corresponding to about 1 mole F to 2 moles DMSO and to 1 mole E to at least 8 moles DMSO. A discontinuity in the CSR function of amylose in the H₂O-DMSO mixed solvent that occurs at 25°C is not observed at 70°C. The CSR function of methyl-β-maltoside exhibits a discontinuity in solvent composition corresponding to mole ratios between 2F–DMSO and 3F–DMSO. Present results indicate that an amylose chain segment may undergo a symmetry change in solvent compositions corresponding to mole ratios between F–DMSO and F–2DMSO. Our CSR measurements of amylose and model compounds in E–DMSO and HMPA–DMSO do not permit us to distinguish between possible changes in amylose chain segment symmetry and solvent interactions that could affect symmetry properties of the glucopyranose ring.     

Influence of solvent molecules on the stereochemical code of glycyl residues in proteins

The Ramachandran steric map and energy diagrams of the glycyl residue are symmetric. A plot of (phi,psi) angles of glycyl residues in 250 nonhomologous and high-resolution protein structures is also largely symmetric. However, there is a clear aberration in the symmetry. Although there is a cluster of points corresponding to the right-handed alpha-helical region, the "equivalent" cluster is clearly shifted to in and around the (phi,psi) values of (90 degrees, 0 degrees ) instead of being centered at the left-handed alpha-helical region of (60 degrees, 40 degrees ). This lack of symmetry exists even in the (phi,psi) distribution of residues from non-alpha-helical regions in proteins. Here we provide an explanation for this observation. An analysis of glycyl conformations in small peptide structures and in "coil" proteins, which are largely devoid of helical and sheet regions, shows that glycyl residues prefer to adopt conformations around (+/-90 degrees, 0 degrees ) instead of right- and left-handed alpha-helical regions. By using theoretical calculations, such conformations are shown to have highest solvent accessibility in a system of two-linked peptide units with glycyl residue at the central C(alpha) atom. This finding is consistent with the observations from 250 nonhomologous protein structures where glycyl residues with conformations close to (+/-90 degrees, 0 degrees ) are seen to have high solvent accessibility. Analysis of a subset of nonhomologous structures with very high resolution (1.5 A or better) shows that water molecules are indeed present at distances suitable for hydrogen bond interaction with glycyl residues possessing conformations close to (+/-90 degrees, 0 degrees ). It is suggested that water molecules play a key role in determining and stabilizing these conformations of glycyl residues and explain the aberration in the symmetry of glycyl conformations in proteins.     

A statistical mechanical model for hydrogen exchange in globular proteins.

We develop a statistical mechanical theory for the mechanism of hydrogen exchange in globular proteins. Using the HP lattice model, we explore how the solvent accessibilities of chain monomers vary as proteins fluctuate from their stable native conformations. The model explains why hydrogen exchange appears to involve two mechanisms under different conditions of protein stability; (1) a “global unfolding” mechanism by which all protons exchange at a similar rate, approaching that of the denatured protein, and (2) a “stable-state” mechanism by which protons exchange at rates that can differ by many orders of magnitude. There has been some controversy about the stable-state mechanism: does exchange take place inside the protein by solvent penetration, or outside the protein by the local unfolding of a subregion? The present model indicates that the stable-state mechanism of exchange occurs through an ensemble of conformations, some of which may bear very little resemblance to the native structure. Although most fluctuations are small-amplitude motions involving solvent penetration or local unfolding, other fluctuations (the conformational distant relatives) can involve much larger transient excursions to completely different chain folds.     

A simple model for polyproline II structure in unfolded states of alanine-based peptides

The striking similarity between observed circular dichroism spectra of nonprolyl homopolymers and that of regular left-handed polyproline II (P_II) helices prompted Tiffany and Krimm to propose in 1968 that unordered peptides and unfolded proteins are built of P_II segments linked by sharp bends. A large body of experimental evidence, accumulated over the past three decades, provides compelling evidence in support of the original hypothesis of Tiffany and Krimm. Of particular interest are the recent experiments of Shi et al. who find significant P_II structure in a short unfolded alanine-based peptide. What is the physical basis for P_II helices in peptide and protein unfolded states? The widely accepted view is that favorable chain-solvent hydrogen bonds lead to a preference for dynamical fluctuations about noncooperative P_II helices in water. Is this preference simply a consequence of hydrogen bonding or is it a manifestation of a more general trend for unfolded states which are appropriately viewed as chains in a good solvent? The prevalence of closely packed interiors in folded proteins suggests that under conditions that favor folding, water—which is a better solvent for itself than for any polypeptide chain—expels the chain from its midst, thereby maximizing chain packing. Implicit in this view is a complementary idea: under conditions that favor unfolding, chain-solvent interactions are preferred and in a so-called good solvent, chain packing density is minimized. In this work we show that minimization of chain packing density leads to preferred fluctuations for short polyalanyl chains around canonical, noncooperative P_II-like conformations. Minimization of chain packing is modeled using a purely repulsive soft-core potential between polypeptide atoms. Details of chain-solvent interactions are ignored. Remarkably, the simple model captures the essential physics behind the preference of short unfolded alanine-based peptides for P_II helices. Our results are based on a detailed analysis of the potential energy landscape which determines the system''s structural and thermodynamic preferences. We use the inherent structure formalism of Stillinger and Weber, according to which the energy landscape is partitioned into basins of attraction around local minima. We find that the landscape for the experimentally studied seven-residue alanine-based peptide is dominated by fluctuations about two noncooperative structures: the left-handed polyproline II helix and its symmetry mate.     

Sterics and solvation winnow accessible conformational space for unfolded proteins

The magnitude of protein conformational space is over-estimated by the traditional random-coil model, in which local steric restrictions arise exclusively from interactions between adjacent chain neighbors. Using a five-state model, we assessed the extent to which steric hindrance and hydrogen bond satisfaction, energetically significant factors, impose additional conformational restrictions on polypeptide chains, beyond adjacent residues. Steric hindrance is repulsive: the distance of closest approach between any two atoms cannot be less than the sum of their van der Waals radii. Hydrogen bond satisfaction is attractive: polar backbone atoms must form hydrogen bonds, either intramolecularly or to solvent water. To gauge the impact of these two factors on the magnitude of conformational space, we systematically enumerated and classified the disfavored conformations that restrict short polyalanyl backbone chains. Applying such restrictions to longer chains, we derived a scaling law to estimate conformational restriction as a function of chain length. Disfavored conformations predicted by the model were tested against experimentally determined structures in the coil library, a non-helix, non-strand subset of the PDB. These disfavored conformations are usually absent from the coil library, and exceptions can be uniformly rationalized.     

Spectroscopic studies on the interaction of bilirubin with symmetrical alkyl diamines

The interactions of symmetrical alkyldiamines with bilirubin-IX α have been examined in dichloromethane and dioxane solutions, by visible region difference sPectroscoPy and florescence methods. In dioxane solutions a clear difference is observed between the comPlexes of the shorter chain diamines (number of sPacer methylene grouPs (n ≤4) ) and the longer chain diamines (n ≥6) . The variations in sPectral features with diamine chain length are less Pronounced in dichloromethane. The sPectroscoPic results are consistent with the occurrence of distinct bilirubin conformations dePending uPon the solvent and the geometry of the interacting recePtor. Based on molecular modelling two conformations are ProPosed. A ‘ridge-tile’ model similar to that observed in crystals is favoured for binding to the longer diamines, while a ‘quasi-cyclic’ structure is Preferred for interaction with the short chain diamines.     

Solvent accessibilities in glycyl, alanyl and seryl dipeptides.

Theoretical studies on glycyl-alanyl and seryl dipeptides were performed to determine the probable backbone and side-group conformations that are preferred for solvent interaction. By following the method of Lee & Richards [(1971) J. Mol. Biol. 55, 379-400], a solute molecule is represented by a set of interlocking spheres of appropriate van der Waals radii assigned to each atom, and a solvent (water) molecule is rolled along the envelope of the van der Waals surface, and the surface accessible to the solvent molecule, and hence the solvent accessibility for a particular conformation of the solute molecule, is computed. From the calculated solvent accessibilities for various conformations, solvation maps for dipeptides were constructed. These solvation maps suggest that the backbone polar atoms could interact with solvent molecules selectively, depending on the backbone conformation. A conformation in the right-handed bridge (zetaR) region is favoured for both solvent interaction and intrachain hydrogen-bonding. Also the backbone side-chain hydrogen-bonding within the same dipeptide fragment in proteins is less favoured than hydrogen-bonding between side chain and water and between side chain and atoms of other residues. Solvent accessibilities suggest that very short distorted alphaR-helical and extended-structural parts may be stabilized via solvent interaction, and this could easily be possible at the surface of the protein molecules, in agreement with protein-crystal data.     

Multiple solution conformations and internal rotations of the decapeptide gramicidin S.

The conformations of every C alpha H-C beta H2 moiety of the peptide gramicidin S are reported. Internal rotation occurs, but distinct preferences for one side chain rotamer, greater than 80%, are found for the D-phenylalanine and ornithine residues. Leucine and valine exhibit more extensive averaging while proline is shown to be at least 90% in the Ramachandran B conformation. The data are consistent with the coexistence of many tertiary conformations of gramicidin S; the statistical weights of the twelve major tertiary conformations consistent with the rotamer populations are reported. The relative statistical weights of the tertiary conformers depend upon temperature and solvent. A comparison of the conclusions from this publication and conformations derived by energy minimization procedures is made. Partial agreement was found, but the calculations have not yet predicted the wealth of coexisting tertiary conformations nor accounted for the subtle effects of solvent. It is proposed that a more complete picture of the conformational dynamics of gramicidin S and other peptides will result from calculations which use as a basis the extensive data reported here.     

Symmetry of multienzyme complexes

A model for studying the symmetry of stable states arising from polyenzymic complex conformations is proposed. A formal scheme of submolecular structure self-assembly, on which the model is based, enables it not only to limit the class of conformations but in some cases to determine the structure of a complex in an unambigous manner. The model is shown in its application to polyenzymic complexes of dehydrogenases of alpha-keto acids.     

Structure refinement of protein model decoys requires accurate side‐chain placement

In this study, the application of temperature‐based replica‐exchange (T‐ReX) simulations for structure refinement of decoys taken from the I‐TASSER dataset was examined. A set of eight nonredundant proteins was investigated using self‐guided Langevin dynamics (SGLD) with a generalized Born implicit solvent model to sample conformational space. For two of the protein test cases, a comparison of the SGLD/T‐ReX method with that of a hybrid explicit/implicit solvent molecular dynamics T‐ReX simulation model is provided. Additionally, the effect of side‐chain placement among the starting decoy structures, using alternative rotamer conformations taken from the SCWRL4 modeling program, was investigated. The simulation results showed that, despite having near‐native backbone conformations among the starting decoys, the determinant of their refinement is side‐chain packing to a level that satisfies a minimum threshold of native contacts to allow efficient excursions toward the downhill refinement regime on the energy landscape. By repacking using SCWRL4 and by applying the RWplus statistical potential for structure identification, the SGLD/T‐ReX simulations achieved refinement to an average of 38% increase in the number of native contacts relative to the original I‐TASSER decoy sets and a 25% reduction in values of C_α root‐mean‐square deviation. The hybrid model succeeded in obtaining a sharper funnel to low‐energy states for a modeled target than the implicit solvent SGLD model; yet, structure identification remained roughly the same. Without meeting a threshold of near‐native packing of side chains, the T‐ReX simulations degrade the accuracy of the decoys, and subsequently, refinement becomes tantamount to the protein folding problem. Proteins 2013. 2012 Published by Wiley Periodicals, Inc.     

NMR studies of a series of dehydrodermorphins

The third and fifth aromatic residues of dermorphin, a potent mu-opioid peptide, and of its N-terminal fragments, from the pentapeptide to the parent heptapeptide amide, have been systematically substituted with Z-dehydrophenylalanine (delta-Phe) and/or Phe to investigate the conformation-activity relationship. The characterization in DMSO-d6 at 500 MHz indicates that, in this solvent, all peptides adopt essentially random, extended conformations, as a consequence of the strong solvation. The chemical shift of the methyl group of D-Ala is influenced by the precise orientation of the side chain of the third residue in a fashion that can be correlated to the mu potency, consistently with our model of mu-receptor. However, the complexes of the pentapeptides with 18-crown-6-ether, when dissolved in chloroform, adopt ordered, folded conformations, a behavior that closely parallels the CD observations in methanol.     

An algorithm for determining the conformation of polypeptide segments in proteins by systematic search

The feasibility of determining the conformation of segments of a polypeptide chain up to six residues in length in globular proteins by means of a systematic search through the possible conformations has been investigated. Trial conformations are generated by using representative sets of phi, psi, and chi angles that have been derived from an examination of the distributions of these angles in refined protein structures. A set of filters based on simple rules that protein structures obey is used to reduce the number of conformations to a manageable total. The most important filters are the maintenance of chain integrity and the avoidance of too-short van der Waals contacts with the rest of the protein and with other portions of the segment under construction. The procedure is intended to be used with approximate models so that allowance is made throughout for errors in the rest of the structure. All possible main chains are first constructed and then all possible side-chain conformations are built onto each of these. The electrostatic energy, including a solvent screening term, and the exposed hydrophobic area are evaluated for each accepted conformation. The method has been tested on two segments of chain in the trypsin like enzyme from Streptomyces griseus. It is found that there is a wide spread of energies among the accepted conformations, and the lowest energy ones have satisfactorily small root mean square deviations from the X-ray structure.     

Protein side chain conformation predictions with an MMGBSA energy function

The prediction of protein side chain conformations from backbone coordinates is an important task in structural biology, with applications in structure prediction and protein design. It is a difficult problem due to its combinatorial nature. We study the performance of an “MMGBSA” energy function, implemented in our protein design program Proteus, which combines molecular mechanics terms, a Generalized Born and Surface Area (GBSA) solvent model, with approximations that make the model pairwise additive. Proteus is not a competitor to specialized side chain prediction programs due to its cost, but it allows protein design applications, where side chain prediction is an important step and MMGBSA an effective energy model. We predict the side chain conformations for 18 proteins. The side chains are first predicted individually, with the rest of the protein in its crystallographic conformation. Next, all side chains are predicted together. The contributions of individual energy terms are evaluated and various parameterizations are compared. We find that the GB and SA terms, with an appropriate choice of the dielectric constant and surface energy coefficients, are beneficial for single side chain predictions. For the prediction of all side chains, however, errors due to the pairwise additive approximation overcome the improvement brought by these terms. We also show the crucial contribution of side chain minimization to alleviate the rigid rotamer approximation. Even without GB and SA terms, we obtain accuracies comparable to SCWRL4, a specialized side chain prediction program. In particular, we obtain a better RMSD than SCWRL4 for core residues (at a higher cost), despite our simpler rotamer library. Proteins 2016; 84:803–819. © 2016 Wiley Periodicals, Inc.     

Modeling loop reorganization free energies of acetylcholinesterase: a comparison of explicit and implicit solvent models

The treatment of hydration effects in protein dynamics simulations varies in model complexity and spans the range from the computationally intensive microscopic evaluation to simple dielectric screening of charge-charge interactions. This paper compares different solvent models applied to the problem of estimating the free-energy difference between two loop conformations in acetylcholinesterase. Molecular dynamics (MD) simulations were used to sample potential energy surfaces of the two basins with solvent treated by means of explicit and implicit methods. Implicit solvent methods studied include the generalized Born (GB) model, atomic solvation potential (ASP), and the distance-dependent dieletric constant. By using the linear response approximation (LRA), the explicit solvent calculations determined a free-energy difference that is in excellent agreement with the experimental estimate, while rescoring the protein conformations with GB or the Poisson equation showed inconsistent and inferior results. While the approach of rescoring conformations from explicit water simulations with implicit solvent models is popular among many applications, it perturbs the energy landscape by changing the solvent contribution to microstates without conformational relaxation, thus leading to non-optimal solvation free energies. Calculations applying MD with a GB solvent model produced results of comparable accuracy as observed with LRA, yet the electrostatic free-energy terms were significantly different due to optimization on a potential energy surface favored by an implicit solvent reaction field. The simpler methods of ASP and the distance-dependent scaling of the dielectric constant both produced considerable distortions in the protein internal free-energy terms and are consequently unreliable.     

The temperature dependence of gramicidin conformational States in octanol

In lipid bilayers and organic solvents, the hydrophobic polypeptide gramicidin adopts a number of different conformations, some of which are capable of conducting monovalent cations across phospholipid membranes. The equilibria between conformations have been shown to be influenced by factors such as lipid chain length, solvent, concentration and salt. In this study, the temperature dependence of the equilibrium mixture of double helical ion-free gramicidin in octanol was examined using circular dichroism spectroscopy.     

Langevin dynamics simulations reveal biologically relevant folds arising from the incorporation of a torsional potential

The conformational behaviour of polymer chains has been examined using Langevin dynamics simulation techniques. Polymer chains were modelled as “beads” undergoing Brownian motion in a defined potential that accounted for stretching, bending and solvation energies. As expected, the competition between chain stiffness and solvent interactions was found to yield standard swollen or collapsed configurations in good or poor solvents, respectively. However, when a torsional term was introduced into the model, additional biologically relevant conformations such as helices, sheets, turns and hairpins naturally arose.     

Solid-state 15N-NMR evidence that gramicidin A can adopt two different backbone conformations in dimyristoylphosphatidylcholine model membrane preparations

Using [15N-Val7]gramicidin A it is shown by solid state 15N-NMR that in dimyristoylphosphatidylcholine model membrane preparations evidence is obtained for two different backbone conformations of gramicidin. One of these conformations is the familiar channel state while a second conformation possesses very different dynamic and structural characteristics. The relative amounts of the conformations depend upon the solvent used to initially codissolve peptide and lipid. Furthermore, by incubation of the samples at modestly elevated temperatures a conversion can be induced from the non-channel to the channel state in a lipid environment.     

Crystal structure of triosephosphate isomerase complexed with 2-phosphoglycolate at 0.83-A resolution

The atomic resolution structure of Leishmania mexicana triosephosphate isomerase complexed with 2-phosphoglycolate shows that this transition state analogue is bound in two conformations. Also for the side chain of the catalytic glutamate, Glu(167), two conformations are observed. In both conformations, a very short hydrogen bond exists between the carboxylate group of the ligand and the catalytic glutamate. The distance between O11 of PGA and Oepsilon2 of Glu(167) is 2.61 and 2.55 A for the major and minor conformations, respectively. In either conformation, Oepsilon1 of Glu(167) is hydrogen-bonded to a water network connecting the side chain with bulk solvent. This network also occurs in two mutually exclusive arrangements. Despite the structural disorder in the active site, the C termini of the beta strands that construct the active site display the least anisotropy compared with the rest of the protein. The loops following these beta strands display various degrees of anisotropy, with the tip of the dimer interface loop 3 having very low anisotropy and the C-terminal region of the active site loop 6 having the highest anisotropy. The pyrrolidine ring of Pro(168) at the N-terminal region of loop 6 is in a strained planar conformation to facilitate loop opening and product release.