Conformational equilibria and free energy profiles for the allosteric transition of the ribose-binding protein

The ribose-binding protein (RBP) is a sugar-binding bacterial periplasmic protein whose function is associated with a large allosteric conformational change from an open to a closed conformation upon binding to ribose. The crystal structures of RBP in open and closed conformations have been solved. It has been hypothesized that the open and closed conformations exist in a dynamic equilibrium in solution, and that sugar binding shifts the population from open conformations to closed conformations. Here, we study by computer simulations the thermodynamic changes that accompany this conformational change, and model the structural changes that accompany the allosteric transition, using umbrella sampling molecular dynamics and the weighted histogram analysis method. The open state is comprised of a diverse ensemble of conformations; the open ribose-free X-ray crystal conformations being representative of this ensemble. The unligated open form of RBP is stabilized by conformational entropy. The simulations predict detectable populations of closed ribose-free conformations in solution. Additional interdomain hydrogen bonds stabilize this state. The predicted shift in equilibrium from the open to the closed state on binding to ribose is in agreement with experiments. This is driven by the energetic stabilization of the closed conformation due to ribose-protein interactions. We also observe a significant population of a hitherto unobserved ribose-bound partially open state. We believe that this state is the one that has been suggested to play a role in the transfer of ribose to the membrane-bound permease complex.     

Stereochemical Criteria for Polypeptide and Protein Chain Conformations: III. Helical and Hydrogen-Bonded Polypeptide Chains

The previous study, for a pair of peptide units, of the conformations which are allowed on the basis of stereochemical criteria of van der Waals contacts has been extended to the analysis of possible conformations of helical polypeptide chains. Computer methods have been developed which select conformations on the basis of both satisfactory interatomic contacts as well as the formation of good intramolecular hydrogen bonds. Such programs have been used to map the allowed dihedral angle pairs (varphi, psi) for helical polypeptide chains. This survey has been made for values of the N-C(a)-C' angle (tau) of 105 degrees , 110 degrees , and 115 degrees , from which the significant influence of this angle in determining allowed helical conformations can be demonstrated. Calculations have also been carried out using potential energy functions for the interaction between nonbonded atoms. The potential energy contour maps obtained in this manner are basically similar to the conformational maps calculated by the first method.     

Conformational study of thyrotrophin-releasing hormone. I. Aspects of importance in the design of novel TRH analogues

The solution conformations of thyrotrophin-releasing hormone (TRH), a biologically-active tripeptide, have been predicted computationally using a method which represents solution effects as a dielectric continuum surrounding the molecule throughout energy minimisation; this is described as the reaction field contribution. The predicted conformations have been compared with previous experimental and theoretical studies. Favoured conformations of TRH under other simulated conditions have also been examined, including the behaviour of the cis isomer, and have been related to possible interactions with intermediates and with specific receptors. The relevance of these predictions to the design of biologically-active analogues of TRH has been analysed.     

Theoretical study of prostaglandin E2 conformation

The theoretical conformational analysis of PGE2 has been performed using semiempirical approach and fixed values of the bond length and valence angles. A set of low-energy conformations was obtained for the alpha-chain and the whole PGE2 molecule. Unlike the PGE1 molecule, PGE2 alpha-chain prefers semifolded conformations. The hairpin structures with the parallel orientation of alpha- and omega-chains are the lowest-energy conformations of PGE2 conformations of PGE1 and PGE2. Sterically similar explain the similar biological activity of the two molecules.     

Comparative conformational analysis of cholesterol and ergosterol by molecular mechanics

A comparative conformational analysis of cholesterol and ergosterol has been carried out using molecular mechanics methods. These studies are aimed at giving a better understanding of the molecular nature of the interaction of these sterols with polyene macrolide antibiotics. Structures of cholesterol and ergosterol determined by X-ray methods have been used as initial geometries of these molecules for force field calculations. The calculation of steric energy has also been made for conformations which do not appear in the crystal. The latter conformers have different conformations of the side chain as well as different conformations of rings A and D. The rotational barriers around bonds C17–C20 and C20–C22 have also been calculated. The results obtained on differences and similarities in the conformations of cholesterol and ergosterol allow us to postulate a mechanism for differential interaction with the antibiotics. The relatively rigid side chain of ergosterol (stretched molecule) in comparison with the flexible side chain of cholesterol (bent molecule), allows better intermolecular contact of the first sterol molecule with a polyene macrolide and in consequence facilitates complex formation involving Van der Waal's forces.     

Helical preferences of alanine,glycine, and aminoisobutyric homopeptides

The stability between helical conformations of homopeptides of alanine, glycine, and aminoisobutyric acid has been studied by means of quantum-mechanical methods. The influence of peptide length on the relative stability between helical conformations has also been analyzed by means of systematic studies for peptides of size up to 11 residues. Finally, the influence of the solvent has been examined by using self-consistent reaction field methods. The results provide a detailed picture of the modulation exerted by these factors on the helical preferences of these peptides. © 1997 Wiley-Liss Inc.     

Comparison of helix-stabilizing effects of alpha,alpha-dialkyl glycines with linear and cycloalkyl side chains

The ability of alpha, alpha-di-n-alkyl glycines with linear and cyclic alkyl side chains to stabilize helical conformations has been compared using a model heptapeptide sequence. The conformations of five synthetic heptapeptides (Boc-Val-Ala-Leu-Xxx-Val-Ala-Leu-OMe, Xxx = Ac8c, Ac7c, Aib, Dpg, and Deg, where Ac8c = 1-aminocyclooctane-1-carboxylic acid, Ac7c = 1-aminocycloheptane-1-carboxylic acid, Aib = alpha-aminoisobutyric acid, Dpg = alpha,alpha-di-n-propyl glycine, Deg = alpha,alpha-di-n-ethyl glycine) have been investigated. In crystals, helical conformations have been demonstrated by x-ray crystallography for the peptides, R-Val-Ala-Leu-Dpg-Val-Ala-Leu-OMe, (R = Boc and acetyl). Solution conformations of the five peptides have been studied by 1H-nmr. In the apolar solvent CDCl3, all five peptides favor helical conformations in which the NH groups of residues 3-7 are shielded from the solvent. Successive NiH<-->Ni + 1H nuclear Overhauser effects over the length of the sequence support a major population of continuous helical conformations. Solvent titration experiments in mixtures of CDCl3/DMSO provide evidence for solvent-dependent conformational transitions that are more pronounced for the Deg and Dpg peptides. Solvent-dependent chemical shift variations and temperature coefficients in DMSO suggest that the conformational distributions in the Deg/Dpg peptides are distinctly different from the Aib/Acnc peptides in a strongly solvating medium. Nuclear Overhauser effects provide additional evidence for the population of extended backbone conformations in the Dpg peptide, while a significant residual population of helical conformations is still detectable in the isomeric Ac7c peptide in DMSO.     

Local structure of cytochrome c from horse heart in solution. Conformational analysis using data of two-dimensional nuclear Overhauser effect spectroscopy]

Using the earlier suggested method the calculation of the backbone conformations of horse heart cytochrome c in oxidized (ferricytochrome c) and reduced (ferrocytochrome c) states has been performed by the two-dimensional nuclear Overhauser effect spectroscopy data. For both protein forms the secondary structure elements have been revealed and the conformations of the irregular polypeptide chain segments have been analysed. The similarity of the secondary structures of ferri- and ferrocytochrome c in solution was established from the comparison of their conformations. Small differences between the conformations of two molecule forms are shown to be localized within the polypeptide chain fragments situated in the spatial structure near the heme crevice. The comparison of the dihedral phi and psi angles in the calculated conformations of horse cytochrome C with the corresponding characteristics of X-ray structures of tuna ferri- and ferrocytochrome c made for the oxidized and reduced protein forms using the quantitative criteria testifies the similarity of their conformations in solution and crystal. In is shown that the conformational changes of the separate amino acid residues which take place as the result of the "solution-to-crystal" transition occur on the surface fragments of protein globule and do not lead to essential alterations of the secondary molecule structure.     

Conformation of glycyl residues in globular proteins

Glycine is unique among the amino acids in view of its symmetric nature. While the overall distribution of glycyl residues in the (phi, psi) plane is near-symmetric, there can be certain preferences for the individual conformations. An analysis of the observed glycyl conformations in 70 proteins has been carried out to find the influence of residues adjoining the glycyl residues. For this purpose, the (phi, psi) plane has been divided into two regions: the region in which phi is negative and the region in which phi is positive. The analysis is done in terms of the number of conformations occurring in these regions. It has been found that while the overall percentage distribution of glycyl residues between the positive and the negative phi regions is 54:46, the distribution shows asymmetry when the examples are sorted out in terms of X-Gly and Gly-Y doublets. The asymmetry becomes more prominent when the data are sorted out into triplets X-Gly-Y. Using the available information, it has been possible to designate 25 triplets as P-predominant and 19 as N-predominant. An examination of P-predominant triplets for possible occurrence in beta-bends having one of the conformations in the positive phi region shows that only 25% are of this nature. Thus, the P-preference of P-predominant triplets is not an outcome of the bend formation alone and must be an inherent property of these triplets.     

Reduced representation model of protein structure prediction: statistical potential and genetic algorithms.

A reduced representation model, which has been described in previous reports, was used to predict the folded structures of proteins from their primary sequences and random starting conformations. The molecular structure of each protein has been reduced to its backbone atoms (with ideal fixed bond lengths and valence angles) and each side chain approximated by a single virtual united-atom. The coordinate variables were the backbone dihedral angles phi and psi. A statistical potential function, which included local and nonlocal interactions and was computed from known protein structures, was used in the structure minimization. A novel approach, employing the concepts of genetic algorithms, has been developed to simultaneously optimize a population of conformations. With the information of primary sequence and the radius of gyration of the crystal structure only, and starting from randomly generated initial conformations, I have been able to fold melittin, a protein of 26 residues, with high computational convergence. The computed structures have a root mean square error of 1.66 A (distance matrix error = 0.99 A) on average to the crystal structure. Similar results for avian pancreatic polypeptide inhibitor, a protein of 36 residues, are obtained. Application of the method to apamin, an 18-residue polypeptide with two disulfide bonds, shows that it folds apamin to native-like conformations with the correct disulfide bonds formed.     

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.     

Conformation characteristics of gamma-glutamyl-containing peptides

Conformational analysis, by the method of atom-atomic potentials, has been carried out for five tripeptides containing gamma-glutamyl bonds and having general formula Glu(gamma)-X-Gly. The spatial structures have been determined and the changes arising on varying the second residue have been analyzed. A comparison of possible conformations and biological activity in respect to a number of enzymes allows to conceive what structural features of these compounds are important for the substrate specificity of the enzymes. In particular, the active site topography has been surmised for glutathione synthetase (EC 6.3.2.3) and gamma-glutamyltranspeptidase (EC 2.3.2.2). The glutathione thiol group has been found to be exposed in all possible conformations that explains its accessibility for various reagents.     

The task of determining the local structure of proteins by Overhauser effect nuclear spectroscopy addressed once more]

The testing of the earlier developed theoretical method for determining the backbone protein conformations (the local structure) on the basis of the two-dimensional nuclear Overhauser effect (NOE) spectroscopy has been fulfilled. The method approval has been carried out by the calculation (based upon spectral NOE parameters) of the local plastocyanin and bovine pancreatic trypsin inhibitor structures followed by the comparison of the received conformational parameters with the X-ray data. The comparison of the molecular conformations in solution and crystal has been implemented for different fragments of the polypeptide chain (beta-structures, alpha-helices, irregular segments) using the mathematical statistics methods. The verification of the "zero" hypothesis about the similarity of phi and psi variation rows which was carried out at the reliability level of 0.99 showed that in both cases there were no systematic deviations of dihedral angles of the compared conformations and that their dispersion differences were statistically indiscernible. It has been concluded that the approved method permits to determine the local structure of the conformationally rigid proteins (or their fragments) at the level close to that which provides the high resolution X-ray analysis.     

江浙蝮蛇毒酸性与碱性磷脂酶A_2溶液构象变化的差示扫描量热法研究

运用差示扫描量热法,在不同ｐＨ值的缓冲溶液内和各种浓度的碱土族氯化物溶液内,研究了来自江浙蝮蛇（ＡｇｋｉｓｔｒｏｄｏｎｈａｌｙｓＰａｌｌａｓ）毒的酸性与碱性磷脂酶外Ａ２（ＰＬＡ２）的热变性过程。得到表征这两种酶溶液构象变化的热力学参数。依据这些参数研究了两者的溶液构象及其变化。在ｐＨ４．５以下,分子净荷正电的这两种酶在溶液中不形成可热致伸展的有序构象;ｐＨ高于４．５时,Ａｓｐ和Ｇｌｕ的侧链羧基以负离子形式存在有利于有序构象的稳定。Ｈｉｓ是决定ＰＬＡ２活力和热稳定性的重要残基。磷酸根离子和这两种酶有结合作用而降低有序构象的热稳定性。碱土族阳离子除和这两种酶结合外,还以依赖于离子强度的方式复杂地影响酶的溶液构象,但其作用不完全是静电性的,是或多或少地随离子的不同而不同的。计算给出酸性ＰＬＡ２的△Ｈｃｄ．     

Conformational studies of -glucans

A study of the effect of linkage on the possible conformations of di-and polysaccharides of α-D -glucose and also the probable intramolecular hydrogen bonds has been made. The differences in the nature of linkage is shown to effect the energetically preferred conformations; (1 → 2) linkages lead only to righthanded helical conformations, (1 → 3) linkages lead to extended as well as both left and righthanded helical conformations; (1 → 4) linkages lead to both right-and lefthanded wide helical conformations. The possible hydrogen bonds between adjacent residues are also dependent on the nature of the linkage. A comparison of the conformational data of α-D -glucans with those of β-D -glucans has indicated that the favored conformations and hydrogen bonds between contiguous residues in the chain are influenced by the configuration at the anomeric carbon atom in all the three types of polysaccharides. From the energy calculations a probable conformation (?M = ?10°, ψM = ?30°, ?N = ?23°, ψN = ?19°) has also been proposed for crystalline mycodextran in conformity with x-ray data. This conformation contains two types of hydrogen bonds between contiguous residues one between 0–2 and 0–3 atoms at (1 → 4) linkage and the other between 0?2 and 0–4 atoms at (1 → 3) linkage in the chain. The conformation of maltose unit (?10°,?30°) that is likely to occur in the crystalline mycodextran coincides with the minimum energy conformation of maltose.     

Rapid generation of a representative ensemble of N-glycan conformations

Glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces where their oligosaccharide moieties are implicated in a wide range of cell-cell and cell-matrix recognition events. Glycans constitute highly flexible molecules. Only a small number of glycan X-ray structures is available for which sufficient electron density for an entire oligosaccharide chain has been observed. An unambiguous structure determination based on NMR-derived geometric constraints alone is often not possible. Time consuming computational approaches such as Monte Carlo calculations and molecular dynamics simulations have been widely used to explore the conformational space accessible to complex carbohydrates. The generation of a comprehensive data base for N-glycan fragments based on long time molecular dynamics simulations is presented. The fragments are chosen in such a way that the effects of branched N-glycan structures are taken into account. The prediction database constitutes the basis of a procedure to generate a complete set of all possible conformations for a given N-glycan. The constructed conformations are ranked according to their energy content. The resulting conformations are in reasonable agreement with experimental data. A web interface has been established (http://www.dkfz.de/spec/glydict/), which enables to input any N-glycan of interest and to receive an ensemble of generated conformations within a few minutes.     

NMR and molecular dynamics studies of tachykinins: conformation of substance P fragment 4-11

The conformation of the C-terminal octapeptide fragment of Substance P (SP4-11, Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) has been investigated by 2D-NMR and MD methods. The octapeptide exists in a blend of conformations. The molecule seems to shuttle between conformations with gamma-bends either at Phe5 or Gly6 or Gln3 or Leu7 and between a nearly extended structure.     

Nanobody stabilization of G protein-coupled receptor conformational states

Remarkable progress has been made in the field of G protein-coupled receptor (GPCR) structural biology during the past four years. Several obstacles to generating diffraction quality crystals of GPCRs have been overcome by combining innovative methods ranging from protein engineering to lipid-based screens and microdiffraction technology. The initial GPCR structures represent energetically stable inactive-state conformations. However, GPCRs signal through different G protein isoforms or G protein-independent effectors upon ligand binding suggesting the existence of multiple ligand-specific active states. These active-state conformations are unstable in the absence of specific cytosolic signaling partners representing new challenges for structural biology. Camelid single chain antibody fragments (nanobodies) show promise for stabilizing active GPCR conformations and as chaperones for crystallogenesis.     

UV resonance Raman investigation of a 3(10)-helical peptide reveals a rough energy landscape

We used UVRRS at 194 and 204 nm excitation to examine the backbone conformation of a 13-residue polypeptide (gp41(659-671)) that has been shown by NMR to predominantly fold into a 3(10)-helix. Examination of the conformation sensitive AmIII(3) region indicates the peptide has significant populations of beta-turn, PPII, 3(10)-helix, and pi-helix-like conformations but little alpha-helix. We estimate that at 1 degree C on average six of the 12 peptide bonds are in folded conformations (predominantly 3(10)- and pi-helix), while the other six are in unfolded (beta-turn/PPII) conformations. The folded and unfolded populations do not change significantly as the temperature is increased from 1 to 60 degrees C, suggesting a unique energy landscape where the folded and unfolded conformations are essentially degenerate in energy and exhibit identical temperature dependences.     

A comparison of the hairpin stability of the palindromic d(CGCG(A/T)4CGCG) oligonucleotides.

The palindromic deoxyribonucleotides 5'-CGCGA-TATCGCG-3' and 5'-CGCGTTAACGCG-3' have been characterized by 1H NMR spectroscopy. The NMR data identified both B-DNA duplex conformations and hairpin conformations, the latter with loop regions consisting of the four central nucleotides. The resonances of the various conformations were assigned by use of two-dimensional NMR methods. The relative stability of the various conformations was investigated as a function of temperature, ionic strength and nucleotide concentration. The duplexes were found to be stabilized at high ionic strength and at low temperature, while the hairpins were stabilized at low ionic strength and at medium temperature. The thermodynamics of the duplex-hairpin and the hairpin-random coil transitions were examined, and compared to the other two oligonucleotide in the palindromic d(CGCG(A/T)4CGCG) oligonucleotide family. The relative stabilities of the duplex conformations with respect to the random coil conformations are similar for the d(CGCGAATTCGCG), d(CGCGATATCGCG) and d(CGCGTATACGCG) oligonucleotides. The duplex conformation of d(CGCGTTAACGCG) is less stable. The hairpin of d(CGCGTTAACGCG) seems also to be less stable relative to the random coil conformation than in the case of the other oligonucleotides at an equal oligonucleotide concentration. A cruciform intermediate between the duplex and hairpin conformations is suggested to explain some discrepancies observed in this work in case of the d(CGCGTTAACGCG) oligonucleotide. This is similar to what has been reported for the d(CGCGTATACGCG) oligonucleotide.