Conformational features of cepacian: the exopolysaccharide produced by clinical strains of Burkholderia cepacia

Conformational energy calculations and molecular dynamics investigations, both in water and in dimethyl sulfoxide, were carried out on the exopolysaccharide cepacian produced by the majority of the clinical strains of Burkholderia cepacia, an opportunistic pathogen causing serious lung infection in patients affected by cystic fibrosis, The investigation was aimed at defining the structural and conformational features, which might be relevant for clarification of the structure-function relationships of the polymer. The molecular dynamics calculations were carried out by Ramachandran-type energy plots of the disaccharides that constitute the polymer repeating unit. The dynamics of an oligomer composed of three repeating units were investigated in water and in Me2SO, a non-aggregating solvent. Analysis of the time persistence of hydrogen bonds showed the presence of a large number of favourable interactions in water, which were less evident in Me2SO. The calculations on the cepacian chain indicated that polymer conformational features in water were affected by the lateral chains, but were also largely dictated by the presence of solvent. Moreover, the large number of intra-chain hydrogen bonds in water disappeared in Me2SO solution, increasing the average dimension of the polymer chains.     

Structure and energy landscape of a photoswitchable peptide: a replica exchange molecular dynamics study

A replica exchange molecular dynamics (REMD) simulation of a bicyclic azobenzene peptide in explicit dimethyl sulfoxide solution is presented in order to characterize the conformational structures and energy landscape of a photoswitchable peptide. It is shown that an enhanced-sampling technique such as the REMD method is essential to obtain a converged conformational sampling of the peptide at room temperature. This is because conventional MD simulations of less than approximately 100-ns length are either trapped in local minima (at 295 K) or-if run at high temperature-do not resemble the room-temperature REMD results. Calculating various nuclear Overhauser effects (NOEs) and (3)J-couplings, a good overall agreement between the REMD simulations and the NMR experiments of Renner et al. (Biopolymers 2000;54:501-514) is found. In particular, the REMD study confirms the general picture drawn by Renner et al. that the trans-isomer of the azobenzene peptide exhibits a well-defined structure, while the cis-isomer is a conformational heterogeneous system; that is, the trans-isomer occurs in 2 well-defined conformers, while the cis-isomer represents an energetically frustrated system that leads to an ensemble of conformational structures. Employing a principal component analysis of the REMD data, the free energy landscape of the systems is studied at various temperatures. The implications for the folding and unfolding pathways of the system are discussed.     

Atomistic simulations of the effects of polyglutamine chain length and solvent quality on conformational equilibria and spontaneous homodimerization

Aggregation of expanded polyglutamine tracts is associated with nine different neurodegenerative diseases, including Huntington's disease. Experiments and computer simulations have demonstrated that monomeric forms of polyglutamine molecules sample heterogeneous sets of collapsed structures in water. The current work focuses on a mechanistic characterization of polyglutamine homodimerization as a function of chain length and temperature. These studies were carried out using molecular simulations based on a recently developed continuum solvation model that was designed for studying conformational and binding equilibria of intrinsically disordered molecules such as polyglutamine systems. The main results are as follows: Polyglutamine molecules form disordered, collapsed globules in aqueous solution. These molecules spontaneously associate at conditions approaching those of typical in vitro experiments for chains of length N ≥ 15. The spontaneity of these homotypic associations increases with increasing chain length and decreases with increasing temperature. Similar and generic driving forces govern both collapse and spontaneous homodimerization of polyglutamine in aqueous milieus. Collapse and dimerization maximize self-interactions and reduce the interface between polyglutamine molecules and the surrounding solvent. Other than these generic considerations, there do not appear to be any specific structural requirements for either chain collapse or chain dimerization; that is, both collapse and dimerization are nonspecific in that disordered globules form disordered dimers. In fact, it is shown that the driving force for intermolecular associations is governed by spontaneous conformational fluctuations within monomeric polyglutamine. These results suggest that polyglutamine aggregation is unlikely to follow a homogeneous nucleation mechanism with the monomer as the critical nucleus. Instead, the results support the formation of disordered, non-β-sheet-like soluble molten oligomers as early intermediates—a proposal that is congruent with recent experimental data.     

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.     

Inhibition of the human erythrocyte calcium pump by dimethyl sulfoxide

The action of dimethyl sulfoxide on the human red cell Ca2+ pump was studied in inside-out vesicles. In a high-K+ medium at pH 7.6, the organic solvent inhibited both Ca2+ transport and ATP hydrolysis. Half-maximal effect was obtained with about 2% (v/v). At or below 10% dimethyl sulfoxide, the inhibition was overcome by adding inorganic phosphate or oxalate. In the absence of organic solvent, Ca2+ efflux from Ca(2+)-loaded vesicles consisted of a slow and a fast component whilst in its presence, there appears additionally a leakage component. The size of the latter depended markedly on dimethyl sulfoxide concentration, being about 3% at that level where Ca2+ uptake was half-maximally inhibited. ATP hydrolysis was more sensitive to dimethyl sulfoxide (10%) when free Ca2+ was increased within the millimolar level than when it was raised within the micromolar range. On the other hand, raising Ca2+ with organic solvent greatly stimulated ATP synthesis through ATP-Pi exchange, without reaching saturation. The results suggest that dimethyl sulfoxide blocks the red cell Ca2+ pump by increasing the affinity of the Ca2+ translocating site at the releasing step. They also show that at high concentrations, this solvent increases Ca2+ permeability.     

Determination of conformational equilibrium of peptides in solution by NMR spectroscopy and theoretical conformational analysis: application to the calibration of mean-field solvation models.

Peptides occur in solution as ensembles of conformations rather than in a fixed conformation. The existing energy functions are usually inadequate to predict the conformational equilibrium in solution, because of failure to account properly for solvation, if the solvent is not considered explicitly (which is usually prohibitively expensive). NMR data are therefore widely incorporated into theoretical conformational analysis. Because of conformational flexibility, restrained molecular dynamics (with restraints derived from NMR data), which is usually applied to determine protein conformation is of limited use in the case of peptides. Instead, (a) the restraints are averaged within predefined time windows during molecular dynamics (MD) simulations (time averaging), (b) multiple-copy MD simulations are carried out and the restraints are averaged over the copies (ensemble averaging), or (c) a representative ensemble of sterically feasible conformations is generated and the weights of the conformations are then fitted so that the computed average observables match the experimental data (weight fitting). All these approaches are briefly discussed in this article. If an adequate force field is used, conformations with large statistical weights obtained from the weight-fitting procedure should also have low energies, which can be implemented in force field calibration. Such a procedure is particularly attractive regarding the parameterization of the solvation energy in nonaqueous solvents, e.g., dimethyl sulfoxide, for which thermodynamic solvation data are scarce. A method for calibration of solvation parameters in dimethyl sulfoxide, which is based on this principle was recently proposed by C. Baysal and H. Meirovitch (Journal of the American Chemical Society, 1998, Vol. 120, pp. 800--812), in which the energy gap between the conformations compatible with NMR data and the alternative conformations is maximized. In this work we propose an alternative method based on the principle that the best-fitting statistical weights of conformations should match the Boltzmann weights computed with the force field applied. Preliminary results obtained using three test peptides of varying conformational mobility: H-Ser(1)-Pro(2)-Lys(3)-Leu(4)-OH, Ac-Tyr(1)-D-Phe(2)-Ser(3)-Pro(4)-Lys(5)-Leu(6)-NH(2), and cyclo(Tyr(1)-D-Phe(2)-Ser(3)-Pro(4)-Lys(5)-Leu(6)) are presented.     

Spectroscopic studies of oligomers containing 2,5-trans furanoid sugar amino acids

Sugar amino acids and their oligomers, known as carbopeptoids, are commonly studied as foldamers. However, study of their conformational preference is often challenging when the adopted conformations are extended and/or disordered. This study is the first to explore the disordered nature of such carbopeptoids by utilizing a family of 2,5-trans carbopeptoids. An array of spectroscopic techniques has been used to investigate the conformational preference of these carbopeptoids. However, using this data alone it has not been possible to assign conformational preference as an ordered extended conformation or as a disordered family of closely related conformations. Computational methods need to be employed to achieve reliable interpretation of the spectroscopic data.     

Stability of hydrolytic enzymes in water-organic solvent systems

The effects of organic solvents on the stabilities of bovine pancreas trypsin, chymotrypsin, carboxypeptidase A and porcine pancreas lipase were studied. Water-miscible solvents (ethanol, acetonitrile, 1,4-dioxane and dimethyl sulfoxide) and water-immiscible solvents (ethyl acetate and toluene) were used in 100 mM phosphate buffer (pH 7.0) or 100 mM Tris/HCl buffer (pH 7.0) in concentrations of 20–80% (v/v). All hydrolytic enzymes studied were inactivated by mixtures containing dimethyl sulfoxide at higher concentrations. Trypsin and carboxypeptidase A resisted solvent mixtures containing acetonitrile, 1,4-dioxane and ethanol. They preserved more than 80% of their starting activities during 20-min incubations. The activities of lipase and chymotrypsin decreased with increasing concentration of water-miscible polar organic solvents, but at higher concentrations (80%) 70–90% of the activity remained. In mixtures with water-immiscible solvents, the decrease in activity of carboxypeptidase A was pronounced. Trypsin and chymotrypsin underwent practically no loss in activity in the presence of toluene or ethyl acetate. In respect of stability, the polar solvent proved to be more favorable for lipase. These results suggest that the conformational stabilities of hydrolytic enzymes are highly dependent on the solvent-protein interactions and the enzyme structure.     

Denaturation of RNA with dimethyl sulfoxide

The denaturation of single-stranded and double-stranded RNA's in solutions with varying proportions of dimethyl sulfoxide has been followed by changes in absorbancy, optical rotation, and—with a double-stranded form of bacteriophage of MS2 RNA— infectivity for bacterial spheroplasts. By these criteria the RNA's studied, including the synthetic polynucleotide rG:rC, are completely denatured at room temperature in high concentrations of this solvent. In lower concentrations, the T_m of the RNA preparation is decreased only slightly as the dimethyl sulfoxide concentration is raised until a critical concentration is reached. The T_m falls sharply with small further increases in dimethyl sulfoxide concentration. Sedimentation studies can be conducted directly in these media. The determination of sedimentation velocity in 99% dimethyl sulfoxide containing 0.001M EDTA provides a reliable estimate of RNA molecular weights.     

Solution conformations of pectin polysaccharides: Determination of chain characteristics by small angle neutron scattering,viscometry, and molecular modeling

The solution behavior of pectin polysaccharides has been investigated by small angle neutron scattering (SANS), viscosimetric, and molecular modeling studies. The samples used in the experimental study were obtained from apple and citrus and had degrees of methylation ranging from 28 to 73%, with a rhamnose content lying between 0.6 and 2.2%. Persistence lengths, derived from intrinsic viscosity measurements, ranged from 59 to 126 Å, whereas those derived by SANS were between 45 and 75 Å. These values correspond to 10–17 monomer units. The modeling simulations were performed for both homogalacturonan itself and homogalacturonan carrying various degrees of rhamnose inserts (rhamnogalacturonan). This required the evaluation of the accessible conformational space for the eight disaccharides that represent the constituent repeating segments of the homogalacturonan and rhamnogalacturonan polysaccharides. For each dimer, complete conformational analysis was accomplished using the flexible residue method of the MM3 molecular mechanics procedure and the results used to access the configurational statistics of representative pectic polysaccharide chains. For homogalacturonan, an extended chain conformation having a persistence length of 135 Å (corresponding to 30 monomers) was predicted. The inclusion of varying amounts of rhamnose units (5–25%) in the model in strict alternating sequence with galacturonate residues (equivalent to the rhamnogalacturonan “hairy region” chains) only slightly reduced the calculated persistence length. The extended overall chain conformation remained relatively unchanged as a consequence of the self-cancellation of the kinking effects of successive paired rhamnose units. © 1996 John Wiley & Sons, Inc.     

The effects of dimethyl sulfoxide on the kinetics of tubulin assembly

The kinetics of tubulin assembly were examined in the absence and presence of dimethyl sulfoxide at 37 degrees C. Inclusion of 1.4 M (10%) dimethyl sulfoxide lowered the critical protein concentration about 8-10-fold, from 9.4 microM in the absence of the organic solvent to 1.1 microM in its presence. This decrease was due solely to an effect on k-, the off rate constant. The on rate constant k+, was essentially unaffected. Another effect of dimethyl sulfoxide was in the nucleation process. The pseudo-first-order rate constant of elongation, kapp (k+[m]), was greatly increased by inclusion of dimethyl sulfoxide. This was due to an increase in the microtubule number concentration, [m]. The microtubules formed in the presence of dimethyl sulfoxide were much shorter than those formed in its absence, accounting for the higher number concentration. The nucleation number, n, was calculated by plots of ln kapp vs. ln c0 or ln t10% vs. ln c0, and the value appeared to be about 4 to 5, although some variability was found. It was shown that a plot of kapp vs. c0 to determine n, is not appropriate because of the inability to distinguish between linear and curved plots in the range of tubulin concentration used in assembly studies.     

Protein flexibility: multiple molecular dynamics simulations of insulin chain B

Multiple molecular dynamics simulations totaling more than 100 ns were performed on chain B of insulin in explicit solvent at 300 K and 400 K. Despite some individual variations, a comparison of the protein dynamics of each simulation showed similar trends and most structures were consistent with NMR experimental values, even at the elevated temperature. The importance of packing interactions in determining the conformational transitions of the protein was observed, sometimes resulting in conformations induced by localized hydrophobic interactions. The high temperature simulation generated a more diverse range of structures with similar elements of secondary structure and populated conformations to the simulations at room temperature. A broad sampling of the conformational space of insulin chain B illustrated a wide range of conformational states with many transitions at room temperature in addition to the conformational states observed experimentally. The T-state conformation associated with insulin activity was consistently present and a possible mechanism of behavior was suggested.     

Solvation Effects on the Conformational Behaviour of Gellan and Calcium Ion Binding to Gellan Double Helices

The contribution of the presence of solvent to the conformations adopted by disaccharide fragments within the repeat unit of gellan have been studied by molecular modelling techniques. Initial conformational energy searches, using a dielectric continuum to represent the solvent, provided starting geometries for a series of molecular dynamics simulations. The solution behaviour from these simulations was subsequently compared to fibre diffraction data of the potassium gellan salt. The present calculations indicate considerable flexibility of the glycosidic linkages, and this is discussed in relation to its effect on gel formation. One of the fragments was solvated with explicit water molecules. These calculations showed the same conformational behaviour as those simulations conducted in implicit solvent.Finally, a series of molecular dynamics (MD) simulations were performed to study the calcium binding to gellan. The results from this clearly showed a well defined binding site for this ion.Abbreviations MM molecular mechanics - MD molecular dynamics     

Exploring the helix-coil transition via all-atom equilibrium ensemble simulations

The ensemble folding of two 21-residue alpha-helical peptides has been studied using all-atom simulations under several variants of the AMBER potential in explicit solvent using a global distributed computing network. Our extensive sampling, orders of magnitude greater than the experimental folding time, results in complete convergence to ensemble equilibrium. This allows for a quantitative assessment of these potentials, including a new variant of the AMBER-99 force field, denoted AMBER-99 phi, which shows improved agreement with experimental kinetic and thermodynamic measurements. From bulk analysis of the simulated AMBER-99 phi equilibrium, we find that the folding landscape is pseudo-two-state, with complexity arising from the broad, shallow character of the "native" and "unfolded" regions of the phase space. Each of these macrostates allows for configurational diffusion among a diverse ensemble of conformational microstates with greatly varying helical content and molecular size. Indeed, the observed structural dynamics are better represented as a conformational diffusion than as a simple exponential process, and equilibrium transition rates spanning several orders of magnitude are reported. After multiple nucleation steps, on average, helix formation proceeds via a kinetic "alignment" phase in which two or more short, low-entropy helical segments form a more ideal, single-helix structure.     

Three-dimensional features of the bacterial polysaccharide (1 → 4)-β-D-glucuronan: A molecular modeling study

The mutant strain M5N1 CS of Rhizobium meliloti produces, in a Rhizobium complete medium supplemented with fructose and sucrose, a partially acetylated homopolymer of D -glucuronic acid residues linked β-(1 → 4). This polysaccharide forms thermoreversible gels with monovalent salts and thermally stable gels with divalent salts. In order to define the different levels of structural characterization, modeling simulations were performed for both the regular (1 → 4)-β-D -glucuronan and the acetylated derivatives. This required the evaluation of the accessible conformational space for the 16 disaccharides. Detailed conformational analysis was accomplished using the flexible residue of the MM3 molecular mechanics procedure and the results were used to access the configurational statistics of representative polysaccharide chains. Within the potential energy surfaces calculated for each disaccharide, several low energy conformers can be identified. When these conformations are extrapolated to regular polysaccharide structures, they generate polymers with right- and left-handed chirality along with a 2-fold axis. This later arrangement (n = 2, h = 5.16 Å) closely corresponds to that derived from a fiber x-ray diffraction investigation. The insertion of acetyl groups induces changes in the helical features of the polymer. As for the simulation of the configurational properties of (1 → 4)-β-D -glucuronan, an extended disordered chain having a persistence length of 105 Å (corresponding to 22 monomers) is predicted. This agrees with previous conclusions derived from solution study. The inclusion of varying amounts of acetyl groups only slightly perturbs the calculated persistence length. © 1998 John Wiley & Sons, Inc. Biopoly 45: 165–175, 1998     

Structure,dynamics, and interactions of jacalin. Insights from molecular dynamics simulations examined in conjunction with results of X‐ray studies

Molecular dynamics simulations have been carried out on all the jacalin–carbohydrate complexes of known structure, models of unliganded molecules derived from the complexes and also models of relevant complexes where X‐ray structures are not available. Results of the simulations and the available crystal structures involving jacalin permit delineation of the relatively rigid and flexible regions of the molecule and the dynamical variability of the hydrogen bonds involved in stabilizing the structure. Local flexibility appears to be related to solvent accessibility. Hydrogen bonds involving side chains and water bridges involving buried water molecules appear to be important in the stabilization of loop structures. The lectin–carbohydrate interactions observed in crystal structures, the average parameters pertaining to them derived from simulations, energetic contribution of the stacking residue estimated from quantum mechanical calculations, and the scatter of the locations of carbohydrate and carbohydrate‐binding residues are consistent with the known thermodynamic parameters of jacalin–carbohydrate interactions. The simulations, along with X‐ray results, provide a fuller picture of carbohydrate binding by jacalin than provided by crystallographic analysis alone. The simulations confirm that in the unliganded structures water molecules tend to occupy the positions occupied by carbohydrate oxygens in the lectin–carbohydrate complexes. Population distributions in simulations of the free lectin, the ligands, and the complexes indicate a combination of conformational selection and induced fit. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Polyethylene glycol-modified enzymes trap water on their surface and exert enzymic activity in organic solvents

Summary Polyethylene glycol-modified enzymes dissolved and had high enzymic activity in organic solvents. A trace amount of water was found to be necessary for the activity. It was reasoned that the amphipathic polymer covalently attached to enzymes kept water molecules around them. This was supported by findings that : (1) high enzymic activity was found in water- immiscible solvents, whereas activity was never observed in water-miscible solvents; (2) enzymic activity was inhibited by increasing the concentration of dimethyl sulfoxide in benzene; (3) activity of lipase was inhibited by a water-miscible alcohol substrate, but was steadily elevated by increasing the concentration of a water-immiscible alcohol substrate; (4) water was not absorbed from benzene solution containing a modified enzyme by molecular sieves, while it was easily absorbed in the presence of a water-miscible organic solvent, dimethyl sulfoxide.     

The "random-coil" state of proteins: comparison of database statistics and molecular simulations.

This study presents a comparison of two models of the random-coil state, one based on statistical distributions from the structural database and the other based on molecular dynamics simulations. The database model relies on the assumption that the random- or statistical-coil state of a particular residue can be described by its conformational distribution in a sufficiently diverse subset of protein structures. The molecular dynamics model is based on distributions from molecular simulations carried out on "dipeptide" models (single residues with N-terminal acetyl and C-terminal N'-methyl amide blocking groups). A comparison of the two models for the residues Ala, Asn, Asp, Gly, and Val indicates that the database distributions are greatly influenced by long-range interactions and dominated by specific recognizable elements of protein structure. In contrast, the limited structural scope of the dipeptide models presents the extreme case of a peptide under the influence of only short-range interactions. The models were evaluated by a comparison of scalar coupling constants calculated from the conformational distributions and compared with experimentally values determined for unstructured peptides. Although the models gave different distributions, there was similar agreement with experiment. This comparison emphasizes the differences and limitations in each model and highlights the difficulty in presenting an accurate picture of the random-coil state. Proteins 1999;36:407- 418.