Solution conformation of proteinase inhibitor IIA from bull seminal plasma by 1H nuclear magnetic resonance and distance geometry

A determination of the solution conformation of the proteinase inhibitor IIA from bull seminal plasma (BUSI IIA) is described. Two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) was used to obtain a list of 202 distance constraints between individually assigned hydrogen atoms of the polypeptide chain, to identify the positions of the three disulfide bridges, and to locate the single cis peptide bond. Supplementary geometric constraints were derived from the vicinal spin-spin couplings and the locations of certain hydrogen bonds, as determined by nuclear magnetic resonance (n.m.r.). Using a new distance geometry program (DISGEO) which is capable of computing all-atom structures for proteins the size of BUSI IIA, five conformers were computed from the NOE distance constraints alone, and another five were computed with the supplementary constraints included. Comparison of the different structures computed from the n.m.r. data among themselves and with the crystal structures of two homologous proteins shows that the global features of the conformation of BUSI IIA (i.e. the overall dimensions of the molecule and the threading of the polypeptide chain) were well-defined by the available n.m.r. data. In the Appendix, we describe a preliminary energy refinement of the structure, which showed that the constraints derived from the n.m.r. data are compatible with a low energy spatial structure.     

Structure and Polymorphism of the Principal Neutralization Site of Thailand HIV-1 Isolate

Abstract

Refining the geometric parameters for the ensemble of conformers, derived earlier in terms of NMR-spectroscopy data for the immunogenic tip of Thailand HIV-1 isolate, was carried out by quantum chemical methods. As a result, (i) the energy characteristics of initial structures were significanly improved, (ii) their relative locations on the scale of formation heats were determined, and (iii) the energy barriers between conformers under study were computed. On the basis of all data obtained, the high resotion 3D structure model, describing the set of stable conformers and containing the biologically active conformation, was proposed for neutralizing epitope of Thailand HIV-1 isolate. The following major conclusions were made based on the analysis of simulated conformations: i) the Gly-Pro-Gly-Gln-Val-Phe stretch forming the immunogenic crown of Thailand HIV-1 isolate exhibits the properties characteristic for metastable oligopeptide that constitutes in solution the dominant structure with other conformations admissible; (ii) three structures out of five NMR-based starting models form the cluster of conformers which adequately describes general conformational features of this functionally important site of gp120; (iii) two structures residing in this cluster are found to be well-ground for implementing the function of immunoreactive conformation of the stretch of interest; (iv) in spite of this observation, the “global” structure which gives rise to inverse γ-turn in the central Gly-Pro-Gly crest of Thailand HIV-1 gp120 is proposed to be the most probable conformation responsible for the formation of viral antigen-antibody complex in particular case under study.     

Structure and polymorphism of the principal neutralization site of Thailand HIV-1 isolate

Refining the geometric parameters for the ensemble of conformers, derived earlier in terms of NMR-spectroscopy data for the immunogenic tip of Thailand HIV-1 isolate, was carried out by quantum chemical methods. As a result, (i) the energy characteristics of initial structures were significantly improved, (ii) their relative locations on the scale of formation heats were determined, and (iii) the energy barriers between conformers under study were computed. On the basis of all data obtained, the high resolution 3D structure model, describing the set of stable conformers and containing the biologically active conformation, was proposed for neutralizing epitope of Thailand HIV-1 isolate. The following major conclusions were made based on the analysis of simulated conformations: i) the Gly-Pro-Gly-Gln-Val-Phe stretch forming the immunogenic crown of Thailand HIV-1 isolate exhibits the properties characteristic for metastable oligopeptide that constitutes in solution the dominant structure with other conformations admissible; (ii) three structures out of five NMR-based starting models form the cluster of conformers which adequately describes general conformational features of this functionally important site of gp120; (iii) two structures residing in this cluster are found to be well-ground for implementing the function of immunoreactive conformation of the stretch of interest; (iv) in spite of this observation, the "global" structure which gives rise to inverse gamma-turn in the central Gly-Pro-Gly crest of Thailand HIV-1 gp120 is proposed to be the most probable conformation responsible for the formation of viral antigen-antibody complex in particular case under study.     

TOUCHSTONE II: a new approach to ab initio protein structure prediction

We have developed a new combined approach for ab initio protein structure prediction. The protein conformation is described as a lattice chain connecting C(alpha) atoms, with attached C(beta) atoms and side-chain centers of mass. The model force field includes various short-range and long-range knowledge-based potentials derived from a statistical analysis of the regularities of protein structures. The combination of these energy terms is optimized through the maximization of correlation for 30 x 60,000 decoys between the root mean square deviation (RMSD) to native and energies, as well as the energy gap between native and the decoy ensemble. To accelerate the conformational search, a newly developed parallel hyperbolic sampling algorithm with a composite movement set is used in the Monte Carlo simulation processes. We exploit this strategy to successfully fold 41/100 small proteins (36 approximately 120 residues) with predicted structures having a RMSD from native below 6.5 A in the top five cluster centroids. To fold larger-size proteins as well as to improve the folding yield of small proteins, we incorporate into the basic force field side-chain contact predictions from our threading program PROSPECTOR where homologous proteins were excluded from the data base. With these threading-based restraints, the program can fold 83/125 test proteins (36 approximately 174 residues) with structures having a RMSD to native below 6.5 A in the top five cluster centroids. This shows the significant improvement of folding by using predicted tertiary restraints, especially when the accuracy of side-chain contact prediction is >20%. For native fold selection, we introduce quantities dependent on the cluster density and the combination of energy and free energy, which show a higher discriminative power to select the native structure than the previously used cluster energy or cluster size, and which can be used in native structure identification in blind simulations. These procedures are readily automated and are being implemented on a genomic scale.     

Mechanical energy contribution of the metatarsophalangeal joint to running and sprinting

Despite the fact that a number of studies have investigated lower extremity energy generation during locomotion, the influence of the metatarsophalangeal (MP) joint remained unknown. The purpose of this study was to determine the relative contribution of the MP joint to the total mechanical energy in running and sprinting. A sagittal plane analysis was performed on data collected from 10 trained male athletes (five runners and five sprinters). The MP moment was assumed to be negligible until the ground reaction force acted distal to the joint. During running, once the ground reaction force crossed the MP joint, the MP moment was plantarflexor for the remainder of ground contact with average peak values of 59.9 Nm. The MP joint moment was plantarflexor throughout the stance phase for sprinting with average peak values of 112.4 Nm. Since the MP joint was dorsiflexing throughout the majority of the stance phase the joint absorbed large amounts of energy, on average 20.9 J during running and 47.8 J during sprinting. A lack of plantarflexion of the MP joint resulted in a lack of energy generation during take-off. Thus, the energy that was absorbed at the joint was dissipated in the shoe and foot structures.     

Kinked structures of isolated nicotinic receptor M2 helices: A molecular dynamics study

The pore-lining M2 helix of the nicotinic acetylcholine receptor exhibits a pronounced kink when the corresponding ion channel is in a closed conformation [N. Unwin (1993) Journal of Molecular Biology, Vol. 229, pp. 1101–1124]. We have performed molecular dynamics simulations of isolated 22-residue M2 helices in order to identify a possible molecular origin of this kink. In order to sample a wide range of conformational space, a simulated annealing protocol was used to generate five initial M2 helix structures, each of which was subsequently used as the basis of 300 ps MD simulations. Two helix sequences (M2α and M2δ) were studied in this manner, resulting in a total often 300 ps trajectories. Kinked helices present in the trajectories were identified and energy minimized to yield a total of five different stable kinked structures. For comparison, a similar molecular dynamics simulation of a Leu₂₃ helix yielded no stable kinked structures. In four of the five kinked helices, the kink was stabilized by H bonds between the helix backbone and polar side-chain atoms. Comparison with data from the literature on site-directed mutagenesis of M2 residues suggests that such polar side-chain to main-chain H bonds may also contribute to kinking of M2 helices in the intact channel protein. © 1994 John Wiley & Sons, Inc.     

Conserved water molecular dynamics of the different X-ray structures of rusticyanin: an unique aquation potentiality of the ligand bonded Cu++ center

The invariant water molecular interaction involving in the Rusticyanin of Thiobacillus ferrooxidans is thought to be important for its molecular complexation with other proteins at differential acidophilic situation. The comparative analysis of the different x-ray, energy minimized, and auto solvated structures of Rusticyanin revealed the presence of five specific invariant bound water molecules (among the approximately 150 water molecules per monomer) in the crystals. The five W 205, W 206, W 112, W 214, and W 221 water molecules (in Rusticyanin PDB code: 1RCY) were seem to be invariant in all the seven structures (PDB codes: 1RCY, 1A3Z, 1A8Z, 1E3O, 1GY1, 1GY2, 2CAL). Among the five conserved water molecules the W 221 (of 1 RCY or the equivalent water molecules in the other oxidized form of Rusticyanin structures) had endowed an interesting coordination potentiality to Cu(+2) ion during the energy minimization. The W 221 was observed to approach toward the tetrahedrally bonded Cu(+2) ion through the opposite (or trans) route of metal-bonded Met 148. This direct water molecular coordination affected the tetrahedral geometry of Cu(+2) to trigonal bipyramidal. Presumably this structural dynamics at the Cu(+2) center could involve in the electron transport process during protein-protein complexation.     

Fold helical proteins by energy minimization in dihedral space and a DFIRE-based statistical energy function

Statistical energy functions are discrete (or stepwise) energy functions that lack van der Waals repulsion. As a result, they are often applied directly to a given structure (native or decoy) without further energy minimization being performed to the structure. However, the full benefit (or hidden defect) of an energy function cannot be revealed without energy minimization. This paper tests a recently developed, all-atom statistical energy function by energy minimization with a fixed secondary helical structure in dihedral space. This is accomplished by combining the statistical energy function based on a distance-scaled finite ideal-gas reference (DFIRE) state with a simple repulsive interaction and an improper torsion energy function. The energy function was used to minimize 2000 random initial structures of 41 small and medium-sized helical proteins in a dihedral space with a fixed helical region. Results indicate that near-native structures for most studied proteins can be obtained by minimization alone. The average minimum root-mean-squared distance (rmsd) from the native structure for all 41 proteins is 4.1 A. The energy function (together with a simple clustering of similar structures) also makes a reasonable selection of near-native structures from minimized structures. The average rmsd value and the average rank for the best structure in the top five is 6.8 A and 2.4, respectively. The accuracy of the structures sampled and the structure selections can be improved significantly with the removal of flexible terminal regions in rmsd calculations and in minimization and with the increase in the number of minimizations. The minimized structures form an excellent decoy set for testing other energy functions because most structures are well-packed with minimum hard-core overlaps with correct hydrophobic/hydrophilic partitioning. They are available online at http://theory.med.buffalo.edu.     

The productive and reproductive biology of flowering plants

Summary The seasonal growth cycles and dry matter partitioning of Heloniopsis orientalis (Thunb.) C. Tanaka (Liliaceae) into its component organs throughout the year were studied and compared with one another in five selected populations at different altitudes in Toyama Prefecture, Japan (i.e., 100, 200, 900, 1,900, and 2,600 m above sea level). In addition, reproductive energy allociation (RA) and reproductive capacities of these five populations were critically analyzed in relation to environmental factors of each habitat.As a result, a remarkable cline in the growth cycle, dry matter allocation as well as significant reproductive characteristics including reproductive capacity and allocation was discovered along the environmental gradients (e.g., the length of the growing season) present as a result of different elevations. There is a clear negative correlation between the reproductive capacities and allocations in these five populations (r=0.9236, P<0.05), i.e., the populations of H. orientalis allocate increasingly greater energy to reproductive activities in response to the increase in elevation, but, on the contrary, show a marked decreasing tendency in reproductive capacity. Likewise, there is a tendency not only of increased energy allocation to total reproductive structures, but also toward producing a single propagule in successively harsher habitats. Specifically, the RA to total reproductive structures at the fruiting stage incrases from 9.97%, 13.03%, 15.25%, 23.06% to 25.52%, and also the relative amount of the energy invested to producing a single propagule increases from 1.000, 1.298, 2.496, 5.512 to 6.079 in response to an increase in elevation.     

Accurate flexible fitting of high-resolution protein structures to small-angle x-ray scattering data using a coarse-grained model with implicit hydration shell

Small-angle x-ray scattering (SAXS) is a powerful technique widely used to explore conformational states and transitions of biomolecular assemblies in solution. For accurate model reconstruction from SAXS data, one promising approach is to flexibly fit a known high-resolution protein structure to low-resolution SAXS data by computer simulations. This is a highly challenging task due to low information content in SAXS data. To meet this challenge, we have developed what we believe to be a novel method based on a coarse-grained (one-bead-per-residue) protein representation and a modified form of the elastic network model that allows large-scale conformational changes while maintaining pseudobonds and secondary structures. Our method optimizes a pseudoenergy that combines the modified elastic-network model energy with a SAXS-fitting score and a collision energy that penalizes steric collisions. Our method uses what we consider a new implicit hydration shell model that accounts for the contribution of hydration shell to SAXS data accurately without explicitly adding waters to the system. We have rigorously validated our method using five test cases with simulated SAXS data and three test cases with experimental SAXS data. Our method has successfully generated high-quality structural models with root mean-squared deviation of 1 ∼ 3 Å from the target structures.     

Conserved Water Molecular Dynamics of the Different X-ray Structures of Rusticyanin: An Unique Aquation Potentiality of the Ligand Bonded Cu++ Center

Abstract

The invariant water molecular interaction involving in the Rusticyanin of Thiobacillus ferrooxidans is thought to be important for its molecular complexation with other proteins at differential acidophilic situation. The comparative analysis of the different x-ray, energy minimized, and auto solvated structures of Rusticyanin revealed the presence of five specific invariant bound water molecules (among the ~ 150 water molecules per monomer) in the crystals. The five W 205, W 206, W 112, W 214, and W 221 water molecules (in Rusticyanin PDB code: 1RCY) were seem to be invariant in all the seven structures (PDB codes: 1RCY, 1A3Z, 1A8Z, 1E3O, 1GY1, 1GY2, 2CAL). Among the five conserved water molecules the W 221 (of 1 RCY or the equivalent water molecules in the other oxidized form of Rusticyanin structures) had endowed an interesting coordination potentiality to Cu⁺² ion during the energy minimization. The W 221 was observed to approach toward the tetrahedrally bonded Cu⁺² ion through the opposite (or trans) route of metal-bonded Met 148. This direct water molecular coordination affected the tetrahedral geometry of Cu⁺² to trigonal bipyramidal. Presumably this structural dynamics at the Cu⁺² center could involve in the electron transport process during protein-protein complexation.     

Use of linear regression model to compare RNA secondary structures

With more and more ribonucleic acid (RNA) secondary structures accumulated, the need for comparing different RNA secondary structures often arises in function prediction and evolutionary analysis. Numerous efficient algorithms were developed for comparing different RNA secondary structures, but challenges remain. In this paper, six new models based on the linear regression model were proposed for the comparison of RNA secondary structures. The proposed models were tested on a mixed data, containing six secondary structures from RNase P RNAs, three secondary structures from SSU rRNA and five secondary structures from 16S ribosomal RNAs. The results have shown the effectiveness of the proposed models. Moreover, the time complexity of our models is favorable by comparing with that of the existing methods which solve the similar problem.     

Molecular characterization of pouched amphistome parasites (Trematoda: Gastrothylacidae) using ribosomal ITS2 sequence and secondary structures

Members of the family Gastrothylacidae (Trematoda: Digenea: Paramphistomata) are parasitic in ruminants throughout Africa and Asia. In north-east India, five species of pouched amphistomes, namely Fischoederius cobboldi, F. elongatus, Gastrothylax crumenifer, Carmyerius spatiosus and Velasquezotrema tripurensis, belonging to this family have been reported so far. In the present study, the molecular phylogeny of these five gastrothylacid species is derived using the second internal transcribed spacer (ITS2) sequence and secondary structure analyses. ITS2 sequence analysis was carried out to see the occurrence of interspecific variations among the species. Phylogenetic analyses were performed for primary sequence data alone as well as the combined sequence-structure information using neighbour-joining and Bayesian approaches. The sequence analysis revealed that there exist considerable interspecific variations among the various gastrothylacid fluke species. In contrast, the inferred secondary structures for the five species using minimum free energy modelling showed structural identities, in conformity with the core four-helix domain structure that has been recently identified as common to almost all eukaryotic taxa. The phylogenetic tree reconstructed using combined sequence-structure data showed a better resolution, as compared to the one using sequence data alone, with the gastrothylacid species forming a monophyletic group that is well separated from members of the other family, Paramphistomidae, of the amphistomid flukes group. The study provides the molecular characterization based on primary sequence data of the rDNA ITS2 region of the gastrothylacid amphistome flukes. Results also demonstrate the phylogenetic utility of the ITS2 sequence-secondary structure data for inferences at higher taxonomic levels.     

Protein NMR Structures Refined without NOE Data

The refinement of low-quality structures is an important challenge in protein structure prediction. Many studies have been conducted on protein structure refinement; the refinement of structures derived from NMR spectroscopy has been especially intensively studied. In this study, we generated flat-bottom distance potential instead of NOE data because NOE data have ambiguity and uncertainty. The potential was derived from distance information from given structures and prevented structural dislocation during the refinement process. A simulated annealing protocol was used to minimize the potential energy of the structure. The protocol was tested on 134 NMR structures in the Protein Data Bank (PDB) that also have X-ray structures. Among them, 50 structures were used as a training set to find the optimal “width” parameter in the flat-bottom distance potential functions. In the validation set (the other 84 structures), most of the 12 quality assessment scores of the refined structures were significantly improved (total score increased from 1.215 to 2.044). Moreover, the secondary structure similarity of the refined structure was improved over that of the original structure. Finally, we demonstrate that the combination of two energy potentials, statistical torsion angle potential (STAP) and the flat-bottom distance potential, can drive the refinement of NMR structures.     

Improved free energy parameters for RNA pseudoknotted secondary structure prediction

Accurate prediction of RNA pseudoknotted secondary structures from the base sequence is a challenging computational problem. Since prediction algorithms rely on thermodynamic energy models to identify low-energy structures, prediction accuracy relies in large part on the quality of free energy change parameters. In this work, we use our earlier constraint generation and Boltzmann likelihood parameter estimation methods to obtain new energy parameters for two energy models for secondary structures with pseudoknots, namely, the Dirks–Pierce (DP) and the Cao–Chen (CC) models. To train our parameters, and also to test their accuracy, we create a large data set of both pseudoknotted and pseudoknot-free secondary structures. In addition to structural data our training data set also includes thermodynamic data, for which experimentally determined free energy changes are available for sequences and their reference structures. When incorporated into the HotKnots prediction algorithm, our new parameters result in significantly improved secondary structure prediction on our test data set. Specifically, the prediction accuracy when using our new parameters improves from 68% to 79% for the DP model, and from 70% to 77% for the CC model.     

G-quadruplex structures of human telomere DNA examined by single molecule FRET and BrG-substitution

The human telomere is known to form the G-quadruplex structure to inhibit the activity of telomerase. Its detailed structure has been of great interest. Recently, two kinds of the parallel-antiparallel hybrid structures have been specified in K(+) solution. However, the G-quadruplex structure is generally thought to be in equilibrium among different structures. Here, we describe the single-pair fluorescence resonance energy transfer (sp-FRET) experiments on telomere samples with bromoguanine (BrG)-substitutions, which control the G-quadruplex structures, at different positions and one without any substitution. The observed FRET distributions were decomposed into five components and the relative population of these components depended on the BrG-substitution positions. In order to consistently explain the variety of conformations, we proposed a novel structural model, the so-called triple-strand-core model. On the basis of this model, the components of the FRET distributions were attributed to the mixed-chair hybrid structures, which were reported recently, and chair-type antiparallel structures, which can be predicted from this model. The FRET efficiencies of these structures were explained in terms of partially broken structures due to steric hindrance and inappropriate capping. This basic model also consistently explains experimental results reported previously. Furthermore, using this model, the folding pathway of the hybrid structures and T-loop formation can be predicted.     

Analysis of the stability of looped-out and stacked-in conformations of an adenine bulge in DNA using a continuum model for solvent and ions

A combination of conformational search, energy minimization, and energetic evaluation using a continuum solvent treatment has been employed to study the stability of various conformations of the DNA fragment d(CGCAGAA)/d(TTCGCG) containing a single adenine bulge. The extra-helical (looped-out) bulge conformation derived from a published x-ray structure and intra-helical (stacked bulge base) model structures partially based on nuclear magnetic resonance (NMR) data were used as start structures for the conformational search. Solvent-dependent contributions to the stability of the conformations were calculated from the solvent exposed molecular surface area and by using the finite difference Poisson-Boltzmann approach. Three classes (I-III) of bulge conformations with calculated low energies can be distinguished. The lowest-energy conformations were found in class I, corresponding to structures with the bulge base stacked between flanking helices, and class II, composed of structures forming a triplet of the bulge base and a flanking base pair. All extra-helical bulge structures, forming class III, were found to be less stable compared with the lowest energy structures of class I and II. The results are consistent with NMR data on an adenine bulge in the same sequence context indicating an intra-helical or triplet bulge conformation in solution. Although the total energies and total electrostatic energies of the low-energy conformations show only relatively modest variations, the energetic contributions to the stability were found to vary significantly among the classes of bulge structures. All intra-helical bulge structures are stabilized by a more favorable Coulomb charge-charge interaction but destabilized by a larger electrostatic reaction field contribution compared with all extra-helical and most triplet bulge structures. Van der Waals packing interactions and nonpolar surface-area-dependent contributions appear to favor triplet class II structures and to a lesser degree also the intra-helical stacked bulge conformations. The large conformational variation found for class III conformers might add a favorable entropic contribution to the stability of the extra-helical bulge form.