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PubMed1996

Explicit solvent models in protein pKa calculations.

Continuum methods for calculation of protein electrostatics treat buried and ordered water molecules by one of two approximations; either the dielectric constant of regions containing ordered water molecules is equal to the bulk solvent dielectric constant, or it is equal to the protein dielectric constant though no fixed atoms are used to represent water molecules. A method for calculating the titration behavior of individual residues in proteins has been tested on models of hen egg white lysozyme containing various numbers of explicit water molecules. Water molecules were included based on hydrogen bonding, solvent accessibility, and/or proximity to titrating groups in the protein. Inclusion of water molecules significantly alters the calculated titration behavior of individual titrating sites, shifting calculated pKa values by up to 0.5 pH unit. Our results suggest that approximately one water molecule within hydrogen-bonding distance of each charged group should be included in protein electrostatics calculations.

Animals↗

PubMed2001

Enthalpic and entropic contributions to the mutational changes in the reduction potential of azurin.

The changes in the reduction potential of Pseudomonas aeruginosa and Alcaligenes denitrificans azurins following point mutations and residue ionizations were factorized into the enthalpic and entropic contributions through variable temperature direct electrochemistry experiments. The effects on the reduction enthalpy due to changes in the first coordination sphere of the copper ion, as in the Met121Gln and Met121His variants of A. denitrificans azurin, insertion of a net charge and alteration in the solvation properties and electrostatic potential in proximity of the metal site, as in the Met44Lys and His35Leu variants of P. aeruginosa azurin, respectively, and proton uptake/release in wild-type and mutated species could invariably be accounted for on the basis of simple coordination chemistry and/or electrostatic considerations. The concomitant changes in reduction entropy were found in general to contribute to the E degrees ' variation to a lesser extent as compared to the enthalpy changes. However, their effects were by no means negligible and in some instances were found to heavily contribute to (or even become the main determinant of) the observed change in reduction potential. Several lines of evidence indicate that the entropic effects are notably influenced by reduction-induced solvent reorganization effects. In particular, protein reduction tends to be favored on entropic grounds with increasing exposure of the copper site to the solvent. Moreover, enthalpy-entropy compensation phenomena are invariably observed when residue mutation or pH-induced conformational changes modify the solvent accessibility of the metal site or alter the H-bonding network in the hydration shell of the molecule. Therefore, in these cases, caution must be used in making predictions of E degrees ' changes simply based on Coulombic or coordination chemistry arguments.

Acid-Base Equilibrium↗

PubMed2001

Synchrotron protein footprinting: a technique to investigate protein-protein interactions.

Traditional approaches for macromolecular structure elucidation, including NMR, crystallography and cryo-EM have made significant progress in defining the structures of protein-protein complexes. A substantial number of macromolecular structures, however, have not been examined with atomic detail due to sample size and heterogeneity, or resolution limitations of the technique; therefore, the general applicability of each method is greatly reduced. Synchrotron footprinting attempts to bridge the gap in these methods by monitoring changes in accessible surface areas of discrete macromolecular moieties. As evidenced by our previous studies on RNA folding and DNA-protein interactions, the three-dimensional structure is probed by examining the reactions of these moieties with hydroxyl radicals generated by synchrotron X-rays. Here we report the application of synchrotron footprinting to the investigation of protein- protein interactions, as the novel technique has been utilized to successfully map the contact sites of gelsolin segment-1 in the gelsolin segment 1/actin complex. Footprinting results demonstrate that phenylalanine 104, located on the actin binding helix of gelsolin segment 1, is protected from hydroxyl radical modification in the presence of actin. This change in reactivity results from the specific protection of gelsolin segment-1, consistent with the substantial decrease in solvent accessibility of F104 upon actin binding, as calculated from the crystal structural of the gelsolin segment 1/actin complex. The results presented here establish synchrotron footprinting as a broadly applicable method to probe structural features of macromolecular complexes that are not amenable to conventional approaches.

Actins↗

PubMed2000

Crystal structure of human ornithine decarboxylase at 2.1 A resolution: structural insights to antizyme binding.

The polyamines spermidine and spermine are ubiquitous and required for cell growth and differentiation in eukaryotes. Ornithine decarboxylase (ODC, EC 4.1.1.17) performs the first step in polyamine biosynthesis, the decarboxylation of ornithine to putrescine. Elevated polyamine levels can lead to down-regulation of ODC activity by enhancing the translation of antizyme mRNA, resulting in subsequent binding of antizyme to ODC monomers which targets ODC for proteolysis by the 26S proteasome. The crystal structure of ornithine decarboxylase from human liver has been determined to 2.1 A resolution by molecular replacement using truncated mouse ODC (Delta425-461) as the search model and refined to a crystallographic R-factor of 21.2% and an R-free value of 28.8%. The human ODC model includes several regions that are disordered in the mouse ODC crystal structure, including one of two C-terminal basal degradation elements that have been demonstrated to independently collaborate with antizyme binding to target ODC for degradation by the 26S proteasome. The crystal structure of human ODC suggests that the C terminus, which contains basal degradation elements necessary for antizyme-induced proteolysis, is not buried by the structural core of homodimeric ODC as previously proposed. Analysis of the solvent-accessible surface area, surface electrostatic potential, and the conservation of primary sequence between human ODC and Trypanosoma brucei ODC provides clues to the identity of potential protein-binding-determinants in the putative antizyme binding element in human ODC.

Amino Acid Sequence↗

PubMed1990

Comparison and accuracy of methodologies employed for analysis of hydropathy, flexibility and secondary structure of proteins.

Various methods have been developed recently for predicting antigenic determinants in a protein by analyzing its hydropathicity, flexibility or secondary structure. The predicted results by these methods were compared directly to the observed structure of the proteins to determine how accurate they were. Regression and correlation of the predicted hydropathicity versus the observed solvent accessibility and the predicted chain flexibility versus actual mobility were determined. Three separate methods of hydropathic analysis had correlation coefficients of 0.52, 0.63 and 0.57 respectively, while a published method for chain flexibility analysis had a 0.19 correlation coefficient. Similarly, agreement between the observed and the predicted secondary structures by two methods was also determined. The limitations of these techniques were clearly defined.

Amino Acid Sequence↗

PubMed2004

A molecular thermodynamic view of DNA-drug interactions: a case study of 25 minor-groove binders.

Developing a molecular view of the thermodynamics of DNA recognition is essential to the design of ligands for regulating gene expression. In a first comprehensive attempt at sketching an atlas of DNA-drug energetics, we present here a detailed thermodynamic view of minor-groove recognition by small molecules via a computational study on 25 DNA-drug complexes. The studies are configured in the MMGBSA (Molecular Mechanics-Generalized Born-Solvent Accessibility) framework at the current state of the art and facilitate a structure-energy component correlation. Analyses were conducted on both energy minimized structures of DNA-drug complexes and molecular dynamics trajectories developed for the purpose of this study. While highlighting the favorable role of packing, shape complementarity, and van der Waals and hydrophobic interactions of the drugs in the minor groove in conformity with experiment, the studies reveal an interesting annihilation of favorable electrostatics by desolvation. Structural modifications attempted on the ligands point to the requisite physico-chemical factors for obtaining improved binding energies. Hydrogen bonds predicted to be important for specificity based on structural considerations do not always turn out to be significant to binding in post facto analyses of molecular dynamics trajectories, which treat thermal averaging, solvent, and counterion effects rigorously. The strength of the hydrogen bonds retained between the DNA and drug during the molecular dynamics simulations is approximately 1kcal/mol. Overall, the study reveals the compensatory nature of the diverse binding free energy components, possible threshold limits for some of these properties, and the availability of a computationally viable free energy methodology which could be of value in drug-design endeavors.

Binding Sites↗

PubMed2006

HARMONY: a server for the assessment of protein structures.

Protein structure validation is an important step in computational modeling and structure determination. Stereochemical assessment of protein structures examine internal parameters such as bond lengths and Ramachandran (varphi,psi) angles. Gross structure prediction methods such as inverse folding procedure and structure determination especially at low resolution can sometimes give rise to models that are incorrect due to assignment of misfolds or mistracing of electron density maps. Such errors are not reflected as strain in internal parameters. HARMONY is a procedure that examines the compatibility between the sequence and the structure of a protein by assigning scores to individual residues and their amino acid exchange patterns after considering their local environments. Local environments are described by the backbone conformation, solvent accessibility and hydrogen bonding patterns. We are now providing HARMONY through a web server such that users can submit their protein structure files and, if required, the alignment of homologous sequences. Scores are mapped on the structure for subsequent examination that is useful to also recognize regions of possible local errors in protein structures. HARMONY server is located at http://caps.ncbs.res.in/harmony/

Algorithms↗

PubMed2005

Reorganization and conformational changes in the reduction of tetraheme cytochromes.

Molecular dynamics simulation (MD) constitutes an alternative to time-consuming experiments for studying conformational changes. We apply MD on a redox system where experimental information exists for the fully oxidized and fully reduced states: tetraheme cytochrome c3. Instead of doing one simulation for each state, we apply 10 4-ns replicas for both states, which provides robust statistics to characterize the redox changes. Besides these long simulations, we perform 120 short ones (50 ps), where an equilibrated oxidized state is perturbed to a reduced state. This allows the application of a nonequilibrium method, the subtraction technique, which makes it possible to characterize the different timescales of conformational changes. Reduction induces conformational changes in the N-terminus and on the loops spanning residues 36-42 and 88-93, which correlate very well with experiments, demonstrating the applicability of this methodology. We also analyze the effect of reduction on hydrogen bonds, solvent accessible surface and bound water, the changes being found to involve the hemes and propionate groups. Redox-induced protonation is also investigated, by protonating the propionates D from hemes I and IV. Although this change in the former does not have major conformational consequences, it induces in the latter conformational changes beyond the ones obtained with reduction.

Computer Simulation↗

PubMed2006

A second paradigm for gene activation in bacteria.

Control of gene expression is key to development and adaptation. Using purified transcription components from bacteria, we employ structural and functional studies in an integrative manner to elaborate a detailed description of an obligatory step, the accessing of the DNA template, in gene expression. Our work focuses on a specialized molecular machinery that utilizes ATP hydrolysis to initiate DNA opening and permits a description of how the events triggered by ATP hydrolysis within a transcriptional activator can lead to DNA opening and transcription. The bacterial EBPs (enhancer binding proteins) that belong to the AAA(+) (ATPases associated with various cellular activities) protein family remodel the RNAP (RNA polymerase) holoenzyme containing the sigma(54) factor and convert the initial, transcriptionally silent promoter complex into a transcriptionally proficient open complex using transactions that reflect the use of ATP hydrolysis to establish different functional states of the EBP. A molecular switch within the model EBP we study [called PspF (phage shock protein F)] is evident, and functions to control the exposure of a solvent-accessible flexible loop that engages directly with the initial RNAP promoter complex. The sigma(54) factor then controls the conformational changes in the RNAP required to form the open promoter complex.

Bacteria↗

PubMed2005

How do side chains orient globally in protein structures?

An angle Omega is defined to serve as a metric for global side-chain orientations, which reflects the orientation of the side chain relative to the radial vector from the center of the protein to an amino acid. The side-chain orientations of buried residues exhibit characteristically different orientations than do exposed residues, in both monomeric and dimeric structures. Overall, buried side chains point mostly inward, whereas surface side chains tend to point outward from the surface. This difference in behavior also correlates well with the residue hydrophobicity; so a global side-chain orientation can be viewed as a direct structural manifestation of hydrophobicity. When various solvent-accessible layers are considered, the behavior is relatively continuous between centrally located and exposed residues. In the case of interfacial residues between subunits, there are statistically significant differences between exposed residues and interface residues for ALA, ARG, ASN, ASP, GLU, HIS, LYS, THR, VAL, MET, PRO, and overall the interface residues have an increased tendency to point inward. Presumably, these substantial differences in orientations of side chains may be a manifestation of hydrophobic forces.

Amino Acids↗

PubMed2002

Analysis of catalytic residues in enzyme active sites.

We present an analysis of the residues directly involved in catalysis in 178 enzyme active sites. Specific criteria were derived to define a catalytic residue, and used to create a catalytic residue dataset, which was then analysed in terms of properties including secondary structure, solvent accessibility, flexibility, conservation, quaternary structure and function. The results indicate the dominance of a small set of amino acid residues in catalysis and give a picture of a general active site environment. It is hoped that this information will provide a better understanding of the molecular mechanisms involved in catalysis and a heuristic basis for predicting catalytic residues in enzymes of unknown function.

Amino Acid Sequence↗

PubMed1997

Classification of the environment of protein residues.

We have studied the classification of the environment of residues within protein structures. Eisenberg's original idea created environmental categories to discriminate between similar residues [Bowie et al., Science (1991), 253, 164-170]. These environments grouped residues based upon their buried surface area, polarity of the surrounding environment, and secondary structure element in which the residue is found. However, Eisenberg's original categories led to incomplete discrimination between residues that only partially substitute for each other. We have expanded on Eisenberg's original idea of environmental categories, by both considering additional contacts in the calculation of the solvent-accessible molecular surface area and by subdividing the environmental plot into regions based upon its theoretical features. Our alternative surface area calculations were used in conjunction with the polarity of the environment of the residue to define a new set of environmental categories. These new categories were able to discriminate between residues such as threonine, valine, and aspartic acid while reflecting the propensity of these residues to substitute for each other.

Amino Acids↗

PubMed2000

Application of multiple sequence alignment profiles to improve protein secondary structure prediction.

The effect of training a neural network secondary structure prediction algorithm with different types of multiple sequence alignment profiles derived from the same sequences, is shown to provide a range of accuracy from 70.5% to 76.4%. The best accuracy of 76.4% (standard deviation 8.4%), is 3.1% (Q(3)) and 4.4% (SOV2) better than the PHD algorithm run on the same set of 406 sequence non-redundant proteins that were not used to train either method. Residues predicted by the new method with a confidence value of 5 or greater, have an average Q(3) accuracy of 84%, and cover 68% of the residues. Relative solvent accessibility based on a two state model, for 25, 5, and 0% accessibility are predicted at 76.2, 79.8, and 86. 6% accuracy respectively. The source of the improvements obtained from training with different representations of the same alignment data are described in detail. The new Jnet prediction method resulting from this study is available in the Jpred secondary structure prediction server, and as a stand-alone computer program from: http://barton.ebi.ac.uk/. Proteins 2000;40:502-511.

Algorithms↗

PubMed1995

Analysis of the loop-helix interaction in bundle motif protein structures.

Molecular dynamics simulations and energy analysis have been carried out to study the structural mobility and stability of the four alpha-helix bundle motifs. The simulation results as well as the X-ray data show that the atomic RMS fluctuation is larger at the loop region for four representative proteins investigated: methemerythrin, cytochrome b-562, cytochrome c', and bovine somatotropin. The loop-loop, helix-helix, and loop-helix interactions are computed for the unfolded and folded proteins. In the folded and solvated protein structures the loop-helix interaction is stronger than the helix-helix interaction, especially in the electrostatic component. But the stabilization energies of both the loop-helix and the helix-helix interactions relative to those of an unfolded structure are of the same order of magnitude. The stabilization due to protein-solvent interaction is greater in the helix region than in the loop region. The percentage of hydrophilic solvent accessible area for the four proteins studied was calculated with the method of Eisenberg and McLachlan. The percentage of the hydrophilic area is greater in the loops than in the helices. A Poisson-Boltzmann calculation shows that the potential from the loops acting on a helix is generally more negative than that from other helices.

Animals↗

PubMed2004

Inter-residue interactions in protein folding and stability.

During the process of protein folding, the amino acid residues along the polypeptide chain interact with each other in a cooperative manner to form the stable native structure. The knowledge about inter-residue interactions in protein structures is very helpful to understand the mechanism of protein folding and stability. In this review, we introduce the classification of inter-residue interactions into short, medium and long range based on a simple geometric approach. The features of these interactions in different structural classes of globular and membrane proteins, and in various folds have been delineated. The development of contact potentials and the application of inter-residue contacts for predicting the structural class and secondary structures of globular proteins, solvent accessibility, fold recognition and ab initio tertiary structure prediction have been evaluated. Further, the relationship between inter-residue contacts and protein-folding rates has been highlighted. Moreover, the importance of inter-residue interactions in protein-folding kinetics and for understanding the stability of proteins has been discussed. In essence, the information gained from the studies on inter-residue interactions provides valuable insights for understanding protein folding and de novo protein design.

Animals↗

PubMed1999

Herbicide-degrading alpha-keto acid-dependent enzyme TfdA: metal coordination environment and mechanistic insights.

TfdA is a non-heme iron enzyme which catalyzes the first step in the oxidative degradation of the widely used herbicide (2, 4-dichlorophenoxy)acetate (2,4-D). Like other alpha-keto acid-dependent enzymes, TfdA utilizes a mononuclear Fe(II) center to activate O(2) and oxidize substrate concomitant with the oxidative decarboxylation of alpha-ketoglutarate (alpha-KG). Spectroscopic analyses of various Cu(II)-substituted and Fe(II)-reconstituted TfdA complexes via electron paramagnetic resonance (EPR), electron spin-echo envelope modulation (ESEEM), and UV-vis spectroscopies have greatly expanded our knowledge of the enzyme's active site. The metal center is coordinated to two histidine residues as indicated by the presence of a five-line pattern in the Cu(II) EPR signal, for which superhyperfine splitting is attributed to two equivalent nitrogen donor atoms from two imidazoles. Furthermore, a comparison of the ESEEM spectra obtained in H(2)O and D(2)O demonstrates that the metal maintains several solvent-accessible sites, a conclusion corroborated by the increase in multiplicity in the EPR superhyperfine splitting observed in the presence of imidazole. Addition of alpha-KG to the Cu-containing enzyme leads to displacement of an equatorial water on copper, as determined by ESEEM analysis. Subsequent addition of 2,4-D leads to the loss of a second water molecule, with retention of a third, axially bound water. In contrast to these results, in Fe(II)-reconstituted TfdA, the cosubstrate alpha-KG chelates to the metal via a C-1 carboxylate oxygen and the alpha-keto oxygen as revealed by characteristic absorption features in the optical spectrum of Fe-TfdA. This binding mode is maintained in the presence of substrate, although the addition of 2,4-D does alter the metal coordination environment, perhaps by creating an O(2)-binding site via solvent displacement. Indeed, loss of solvent to generate an open binding site upon the addition of substrate has also been suggested for the alpha-keto acid-dependent enzyme clavaminate synthase 2 [Zhou et al. (1998) J. Am. Chem. Soc. 120, 13539-13540]. Nitrosyl adducts of various Fe-TfdA complexes have also been investigated by optical and EPR spectroscopy. Of special interest is the tightly bound NO complex of Fe-TfdA.(alpha-KG).(2,4-D), which may represent an accurate model of the initial oxygen-bound species.

2,4-Dichlorophenoxyacetic Acid↗

PubMed2003

Protein side-chain rearrangement in regions of point mutations.

A major problem in predicting amino acid side-chain rearrangements following point mutations is the potentially large search space. We analyzed a nonredundant data set of 393 Protein Data Bank protein pairs, each consisting of structures differing in one amino acid, to determine the number of residues changing conformation in the region of mutation. In 91-95% of cases, two or fewer residues underwent side-chain conformational change. If mutation sites with backbone displacements were excluded, the number increased to 97%. The majority of rearrangements (over 60%) were due to the inherent flexibility of side-chains, as derived from analysis of a control set of protein subunits whose crystal structures were determined more than once. Different amino acids demonstrated different degrees of flexibility near mutation sites. Large polar or charged residues, and serine, are more flexible, while the aromatic amino acids, and cysteine, are less so. This pattern is common to the inherent side-chain flexibility, as well as the increased flexibility at ligand binding sites and mutation sites. The probability for conformational change was correlated with B-factor, frequency of the side-chain conformation in proteins and solvent accessibility. The last trend was stronger for aromatic and hydrophilic residues than for hydrophobic ones. We conclude that the search space for predicting side-chain conformations in the region of mutation can be effectively restricted. However, the overall ability to predict a particular side-chain conformation, or to check predictions according to individual existing structures, is limited. These findings may be useful in deriving empirical rules for modeling side-chain conformations.

Binding Sites↗

PubMed2001

Probing subtle differences in the hydrogen exchange behavior of variants of the human alpha-lactalbumin molten globule using mass spectrometry.

The hydrogen-exchange behavior of the low-pH molten globule of human alpha-lactalbumin, containing all four disulfides, has been examined and compared with that of a single disulfide variant, [28-111] alpha-lactalbumin, and of a series of proline variants of [28-111] alpha-lactalbumin. The small differences in hydrogen-exchange protection exhibited by these partially folded species were compared by mixing two or more proteins and monitoring their exchange simultaneously using mass spectrometry. The effect of single proline mutations within each alpha-domain helix on hydrogen-exchange protection has been investigated using six proline variants of [28-111] alpha-lactalbumin, L11P, L12P, M30P, I95P, K108P and Q117P. The results show that proline mutations in the A, B, C and D alpha-helices lead to a loss of hydrogen-exchange protection for residues in the local helix without perturbing hydrogen-exchange protection in other regions of the protein. Thus, local unfolding of the A, B, C and D helices does not significantly alter the packing and solvent accessibility of other regions of the molten globule. By contrast, introduction of a proline residue in the C-terminal 3(10) helix produces a larger and more widespread loss of hydrogen-exchange protection, demonstrating that longer-range perturbations of the molten globule have occurred. Thus, residues in this C-terminal region must be involved in contacts that are critical for the stabilisation of the compact molten globule structure.

Binding Sites↗