Residue solvent accessibilities in the unfolded polypeptide chain.

The difference of solvent accessibilities in the native and unfolded states of the protein is used as a measure of the hydrophobic contribution to the free energy of folding. We present a new approximation of amino acids solvent accessibilities in the unfolded state based on the 1-ns molecular dynamics simulation of Ala-X-Ala tripeptides at a temperature of 368 K. The standard accessibility values averaged from the molecular dynamics study are significantly lower from those previously obtained by considering only selected conformations of Ala-X-Ala tripeptides.     

3 Nsec molecular dynamics simulation of the protein ubiquitin and comparison with X-ray crystal and solution NMR structures.

Mainly due to computational limitations, past protein molecular dynamics simulations have rarely been extended to 300 psec; we are not aware of any published results beyond 350 psec. The present work compares a 3000 psec simulation of the protein ubiquitin with the available x-ray crystallographic and solution NMR structures. Aside from experimental structure availability, ubiquitin was studied because of its relatively small size (76 amino acids) and lack of disulfide bridges. An implicit solvent model was used except for explicit treatment of waters of crystallization. We found that the simulated average structure retains most of the character of the starting x-ray crystal structure. In two highly surface accessible regions, the simulation was not in agreement with the x-ray structure. In addition, there are six backbone-backbone hydrogen bonds that are in conflict between the solution NMR and x-ray crystallographic structures; two are bonds that the NMR does not locate, and four are ones that the two methods disagree upon the donor. Concerning these six backbone-backbone hydrogen bonds, the present simulation agrees with the solution NMR structure in five out-of-the six cases, in that if a hydrogen bond is present in the x-ray structure and not in the NMR structure, the bond breaks within 700 psec. Of the two hydrogen bonds that are found in the NMR structure and not in the x-ray structure, one forms at 1400 psec and the other forms rarely. The present results suggest that relatively long molecular dynamics simulations, that use protein x-ray crystal coordinates for the starting structure and a computationally efficient solvent representation, may be used to gain an understanding of conformational and dynamic differences between the solid-crystal and dilute-solution states.     

Application of Dirichlet process mixture model to the identification of spin systems in protein NMR spectra

Protein structure determination using NMR is dependent on experimentally acquired distance restraints. Often, however, an insufficient number of these restraints are available for determining a protein’s correct fold, much less its detailed three-dimensional structure. In consideration of this problem, we propose a simple means to acquire supplemental structural restraints from protein surface accessibilities using solvent saturation transfer to proteins (SSTP), based on the principles of paramagnetic chemical-exchange saturation transfer. Here, we demonstrate the utility of SSTP in structure calculations of two proteins, TSG101 and ubiquitin. The observed SSTP was found to be directly proportional to solvent accessibility. Since SSTP does not involve the direct excitation of water, which compromises the analysis of protein protons entangled in the breadth of the water resonance, it has an advantage over conventional water-based magnetization transfers. Inclusion of structural restraints derived from SSTP improved both the precision and accuracy of the final protein structures in comparison to those determined by traditional approaches, when using minimal amounts of additional structural data. Furthermore, we show that SSTP can detect weak protein–protein interactions which are unobservable by chemical shift perturbations.     

The use of NMR chemical shifts to analyse the MD trajectories: simulation of bovine pancreatic trypsin inhibitor dynamics in water as a test case for solvent influences.

In this paper the NMR secondary chemical shifts, that are estimated from a set of 3D-structures, are compared with the observed ones to appraise the behaviour of a known x-ray diffraction structure (of the bovine pancreatic trypsin inhibitor protein) when various molecular dynamics are applied. The results of a 200 ps molecular dynamics under various conditions are analysed and different ways to modify the molecular dynamics are considered. With the purpose of avoiding the time-consuming explicit representation of the solvent (water) molecules, an attempt was made to understand the role of the solvent and to develop an implicit representation, which may be refined. A simulation of hydrophobic effects in an aqueous environment is also proposed which seems to provide a better approximation of the observed solution structure of the protein.     

Structural refinement of membrane proteins by restrained molecular dynamics and solvent accessibility data

We present an approach for incorporating solvent accessibility data from electron paramagnetic resonance experiments in the structural refinement of membrane proteins through restrained molecular dynamics simulations. The restraints have been parameterized from oxygen (ΠO₂) and nickel-ethylenediaminediacetic acid (ΠNiEdda) collision frequencies, as indicators of lipid or aqueous exposed spin-label sites. These are enforced through interactions between a pseudoatom representation of the covalently attached Nitroxide spin-label and virtual “solvent” particles corresponding to O₂ and NiEdda in the surrounding environment. Interactions were computed using an empirical potential function, where the parameters have been optimized to account for the different accessibilities of the spin-label pseudoatoms to the surrounding environment. This approach, “pseudoatom-driven solvent accessibility refinement”, was validated by refolding distorted conformations of the Streptomyces lividans potassium channel (KcsA), corresponding to a range of 2-30 Å root mean-square deviations away from the native structure. Molecular dynamics simulations based on up to 58 electron paramagnetic resonance restraints derived from spin-label mutants were able to converge toward the native structure within 1-3 Å root mean-square deviations with minimal computational cost. The use of energy-based ranking and structure similarity clustering as selection criteria helped in the convergence and identification of correctly folded structures from a large number of simulations. This approach can be applied to a variety of integral membrane protein systems, regardless of oligomeric state, and should be particularly useful in calculating conformational changes from a known reference crystal structure.     

NMR studies of protein hydration and TEMPOL accessibility

Understanding the mechanisms of the interaction between a protein surface and its outer molecular environment is of primary relevance for the rational design of new drugs and engineered proteins. Protein surface accessibility is emerging as a new dimension of Structural Biology, since NMR methods have been developed to follow how molecules, even those different from physiological ligands, preferentially approach specific regions of the protein surface. Hen egg-white lysozyme, a paradigmatic example of the state of the art of protein structure and dynamics, has been selected as a model system to study protein surface accessibility. Bound water and soluble spin-labels have been used to investigate the interaction of this enzyme, both free and bound to the inhibitor (NAG)(3), with its molecular environment. No tightly bound water molecules were found inside the enzyme active site, which, conversely, appeared as the most exposed to visits from the soluble paramagnetic probe TEMPOL. From the presented set of data, an integrated view of lysozyme surface accessibility towards water and TEMPOL molecules is obtained.     

The yeast mitochondrial citrate transport protein: determination of secondary structure and solvent accessibility of transmembrane domain IV using site-directed spin labeling

To explore the spatial organization and functional dynamics of the citrate transport protein (CTP), a nitroxide scan was carried out along 22 consecutive residues within the fourth transmembrane domain (TMDIV). This domain has been implicated as being of unique importance to the CTP mechanism due to (i) the presence of two intramembranous positive charges that are essential for CTP function and (ii) the existence of a transmembrane aqueous surface within this domain which likely corresponds to a portion of the citrate translocation pathway. The sequence-specific variation in the mobilities of the introduced nitroxides and their accessibilities to molecular O(2) reveal an alpha-helical conformation along the sequence. The accessibilities to NiEDDA are out of phase with accessibilites to O(2), indicating that one face of the helix is solvated by the lipid bilayer while the other is solvated by an aqueous environment. A gradient of NiEDDA accessibility is observed along the helix surface facing the aqueous phase, and the EPR spectral line shapes at these sites indicate considerable motional restriction. In the context of the model where TMDIV lines the translocation pathway, these data suggest a barrier to passive diffusion through the pathway. This paper reports the first use of site-directed spin labeling to study mitochondrial transporter structure.     

Mechanism of surface peptide proton exchange in bovine pancreatic trypsin inhibitor. Salt effects and O-protonation

The acid-catalyzed hydrogen exchange rate constants kH, and the base-catalyzed rate constants kOH, have been determined (in the preceding paper) for the 25 most rapidly exchanging NH groups of bovine pancreatic trypsin inhibitor. Most of these NH groups are at the protein-solvent interface. The correlation of kH, but not kOH, with the static accessibility and hydrogen bonding of the peptide carbonyl O atom indicates that the mechanism of acid catalysis in proteins involves O-protonation. Agreement between the ionic strength dependence observed for kH and kOH and the ionic strength dependence calculated for an O-protonation mechanism supports this conclusion. N-protonation for acid catalysis, as well as N-deprotonation for base catalysis, have traditionally been assumed in the mechanism of the chemical step in peptide amide proton exchange. A preference for the alternative O-protonation mechanism has far-reaching implications in the interpretation of protein hydrogen exchange kinetics. With an O-protonation mechanism, acid-catalyzed rates of surface NH groups are primarily a function of the average solvent accessibility of the carbonyl O atoms in the dynamic solution structure, while base-catalyzed rates of surface NH groups measure solvent accessibility of the peptide N. The relative dynamic accessibilities of peptide O atoms, as measured by relative values of kH (corrected for electrostatic effects), correlate with O static accessibilities in the crystal structure. A lower correlation of static accessibility of N atoms with kOH is observed for surface NH groups in peptide groups in which the carbonyl O is not hydrogen bonded. For some surface NH groups, the observed pH of minimum rate, pHmin, deviates widely from the pHmin of model compounds. This is explained as the combined result of electrostatic effects and of the differences in accessibility of the carbonyl O and N atoms that result in a change in the relative values of kH and kOH as compared to those of model peptides. A mechanism whereby exchange of interior sites is catalyzed by interactions of catalysis ions with protein surface atoms via charge transfer is suggested.     

Solvent accessibilities in glycyl, alanyl and seryl dipeptides.

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

MD and NMR studies of alpha-bungarotoxin surface accessibility

Protein surface accessibility represents a dimension of structural biology which has not been discussed in details so far, in spite of its fundamental role in controlling the molecular recognition process. In the present report the surface accessibility of alpha-bungarotoxin, a small and well characterized protein, has been investigated by analyzing its interaction with solvent and paramagnetic molecules in an integrated way. The presence of strong hydration sites, identified by a combined analysis of MD simulation and NMR results, seems to prevent the access of Gd(III)DTPA-BMA to the protein surface. On the contrary, the limited hydration of the alpha-bungarotoxin active site favors frequent encounters between the paramagnetic probe and the protein in the latter region. All the data obtained here for alpha-bungarotoxin suggest that shape and stability of the solvation shell control its surface accessibility and, hence, intermolecular interactions in a way which could be common to many other proteins.     

Prediction of protein relative solvent accessibility with a two-stage SVM approach

Information on relative solvent accessibility (RSA) of amino acid residues in proteins provides valuable clues to the prediction of protein structure and function. A two-stage approach with support vector machines (SVMs) is proposed, where an SVM predictor is introduced to the output of the single-stage SVM approach to take into account the contextual relationships among solvent accessibilities for the prediction. By using the position-specific scoring matrices (PSSMs) generated by PSI-BLAST, the two-stage SVM approach achieves accuracies up to 90.4% and 90.2% on the Manesh data set of 215 protein structures and the RS126 data set of 126 nonhomologous globular proteins, respectively, which are better than the highest published scores on both data sets to date. A Web server for protein RSA prediction using a two-stage SVM method has been developed and is available (http://birc.ntu.edu.sg/~pas0186457/rsa.html).     

The interpretation of protein structures: estimation of static accessibility

A program is described for drawing the van der Waal's surface of a protein molecule. An extension of the program permits the accessibility of atoms, or groups of atoms, to solvent or solute molecules of specified size to be quantitatively assessed. As defined in this study, the accessibility is proportional to surface area. The accessibility of all atoms in the twenty common amino acids in model tripeptides of the type Ala-X-Ala are given for defined conformation. The accessibilities are also given for all atoms in ribonuclease-S, lysozyme and myogoblin. Internal cavities are defined and discussed. Various summaries of these data are provided. Forty to fifty per cent of the surface area of each protein is occupied by non-polar atoms. The actual numerical results are sensitive to the values chosen for the van der Waal's radii of the various groups. Since there is uncertainty over the correct values for these radii, the derived numbers should only be used as a qualitative guide at this stage.     

Cryo-Cooling Effect on DHFR Crystal Studied by Replica-Exchange Molecular Dynamics Simulations

Cryo-cooling is routinely performed before x-ray diffraction image collection to reduce the damage to crystals due to ionizing radiation. It has been suggested that although backbone structures are usually very similar between room temperature and cryo-temperature, cryo-cooling may hamper biologically relevant dynamics. In this study, the crystal of Escherichia coli dihydrofolate reductase is studied with replica-exchange molecular dynamics simulation, and the results are compared with the crystal structure determined at cryo-temperature and room temperature with the time-averaged ensemble method. Although temperature dependence of unit cell compaction and root mean-square fluctuation of Cα is found in accord with experiment, it is found that the protein structure at low temperature can be more heterogeneous than the ensemble of structures reported by using the time-averaged ensemble method, encouraging further development of the time-averaged ensemble method and indicating that data should be examined carefully to avoid overinterpretation of one average structure.     

Tryptophanyl substitutions in apomyoglobin affect conformation and dynamic properties of AGH subdomain

The solvent accessibilities to the tryptophanyl microenvironments of wild type sperm whale apomyoglobin (apoMb) and two mutants (W7F and W14F) containing a single tryptophan are measured by fluorescence quenching studies. The results are compared to those relative to horse apoMb. In the wild type sperm whale protein, no difference is noticed in the solvent accessibility of the two indole residues, as documented by the values of the Stern-Volmer constants. By contrast, the two tryptophan residues of horse apoMb are exposed to the solvent in a different way, thus indicating that some local conformational differences exist between the two homologous proteins in solution. The single W --> F substitution at either position 7 or 14 determines local conformational changes that increase the accessibility of the remaining indole residue but do not affect the overall architecture of the protein molecule.     

Molecular Dynamics Simulations of Calcium-Free Calmodulin in Solution

Abstract

A 4-ns molecular dynamics simulation of calcium-free calmodulin in solution has been performed, using Ewald summation to treat electrostatic interactions. Our simulation results were mostly consistent with solution experimental studies, including NMR, fluorescence and x-ray scattering. The secondary structures within the N- and C-terminal domains were conserved in the simulation, with trajectory structures similar to the NMR-derived model structure 1CFD. However, the relative orientations of the domains, for which there are no NMR restraints, differed in details between the simulation and the 1CFD model. The most interesting information provided by the simulations is that the dynamics of calcium-free calmod- ulin in solution is dominated by slow rigid body reorientations of the domains. The interdomain distance fluctuated between 29 and 39 Å, and interdomain orientation angle, defined as the pseudo-dihedral formed by the four calcium binding sites, varied between ?2° and 108°. Similarly, the domain linker region also exhibited significant fluctuations, with its length varying in the 34–45 Å range and its bend angle in the 10–100° range. The simulations are in accord with fluorescence results suggesting that calcium-free calmodulin is more compact and more flexible than the calcium activated form. Surprisingly, quite similar solvent accessibilities of the hydrophobic patches were seen in the calcium-free trajectory described in this work and previously generated calcium-loaded calmodulin simulations. Thus, our simulations suggest a reexamination of the standard model of the structural change of calmodulin upon calcium binding, involving exposure of the hydrophobic patches to solvent.     

Molecular dynamics simulations of calcium-free calmodulin in solution

A 4-ns molecular dynamics simulation of calcium-free calmodulin in solution has been performed, using Ewald summation to treat electrostatic interactions. Our simulation results were mostly consistent with solution experimental studies, including NMR, fluorescence and x-ray scattering. The secondary structures within the N- and C-terminal domains were conserved in the simulation, with trajectory structures similar to the NMR-derived model structure 1CFD. However, the relative orientations of the domains, for which there are no NMR restraints, differed in details between the simulation and the 1CFD model. The most interesting information provided by the simulations is that the dynamics of calcium-free calmodulin in solution is dominated by slow rigid body reorientations of the domains. The interdomain distance fluctuated between 29 and 39 A, and interdomain orientation angle, defined as the pseudo-dihedral formed by the four calcium binding sites, varied between -2 degrees and 108 degrees. Similarly, the domain linker region also exhibited significant fluctuations, with its length varying in the 34-45 A range and its bend angle in the 10-100 degrees range. The simulations are in accord with fluorescence results suggesting that calcium-free calmodulin is more compact and more flexible than the calcium activated form. Surprisingly, quite similar solvent accessibilities of the hydrophobic patches were seen in the calcium-free trajectory described in this work and previously generated calcium-loaded calmodulin simulations. Thus, our simulations suggest a reexamination of the standard model of the structural change of calmodulin upon calcium binding, involving exposure of the hydrophobic patches to solvent.     

Average assignment method for predicting the stability of protein mutants

Prediction of protein stability upon amino acid substitutions is an important problem in molecular biology and it will be helpful for designing stable mutants. In this work, we have analyzed the stability of protein mutants using three different data sets of 1791, 1396, and 2204 mutants, respectively, for thermal stability (DeltaTm), free energy change due to thermal (DeltaDeltaG), and denaturant denaturations (DeltaDeltaGH2O), obtained from the ProTherm database. We have classified the mutants into 380 possible substitutions and assigned the stability of each mutant using the information obtained with similar type of mutations. We observed that this assignment could distinguish the stabilizing and destabilizing mutants to an accuracy of 70-80% at different measures of stability. Further, we have classified the mutants based on secondary structure and solvent accessibility (ASA) and observed that the classification significantly improved the accuracy of prediction. The classification of mutants based on helix, strand, and coil distinguished the stabilizing/destabilizing mutants at an average accuracy of 82% and the correlation is 0.56; information about the location of residues at the interior, partially buried, and surface regions of a protein correctly identified the stabilizing/destabilizing residues at an average accuracy of 81% and the correlation is 0.59. The nine subclassifications based on three secondary structures and solvent accessibilities improved the accuracy of assigning stabilizing/destabilizing mutants to an accuracy of 84-89% for the three data sets. Further, the present method is able to predict the free energy change (DeltaDeltaG) upon mutations within a deviation of 0.64 kcal/mol. We suggest that this method could be used for predicting the stability of protein mutants.     

Characterization of Protein–Protein Interfaces

We analyze the characteristics of protein–protein interfaces using the largest datasets available from the Protein Data Bank (PDB). We start with a comparison of interfaces with protein cores and non-interface surfaces. The results show that interfaces differ from protein cores and non-interface surfaces in residue composition, sequence entropy, and secondary structure. Since interfaces, protein cores, and non-interface surfaces have different solvent accessibilities, it is important to investigate whether the observed differences are due to the differences in solvent accessibility or differences in functionality. We separate out the effect of solvent accessibility by comparing interfaces with a set of residues having the same solvent accessibility as the interfaces. This strategy reveals residue distribution propensities that are not observable by comparing interfaces with protein cores and non-interface surfaces. Our conclusions are that there are larger numbers of hydrophobic residues, particularly aromatic residues, in interfaces, and the interactions apparently favored in interfaces include the opposite charge pairs and hydrophobic pairs. Surprisingly, Pro-Trp pairs are over represented in interfaces, presumably because of favorable geometries. The analysis is repeated using three datasets having different constraints on sequence similarity and structure quality. Consistent results are obtained across these datasets. We have also investigated separately the characteristics of heteromeric interfaces and homomeric interfaces.     

Parametrisation of time-averaged distance restraints in MD simulations

Summary Time-averaged restraints in molecular dynamics simulations offer a means to account for the averaging that is implicit in NMR spectroscopic data. We present a systematic investigation of the parameters which characterise time-averaged distance restraints. Using previously published data for a small protein, chymotrypsin inhibitor 2, we identify conditions which can lead to undesirable heating or which grossly distort the dynamics of the system.Abbreviations NOE nuclear Overhauser effect - MD molecular dynamics - CI-2 chymotrypsin inhibitor 2