FIRE: predicting the spatial proximity of protein residues from 3D NOESY–HSQC

 We address the problem of the prediction of residue spatial proximity in a protein, through the automatic processing of a 3D ¹⁵N NOESY–HSQC. The spatial distance between residues is estimated from a spectral match value calculated using a comparison of the resonances involving the amide hydrogens. The method is shown to provide a good estimation of a large number of residue spatial proximities, in the case of two experimental 3D spectra, recorded on proteins of α and β secondary structures. It is tested on simulated data sets against the protein size, secondary structure and the quality of the signal. More than 70% of the sequential assignment is correctly predicted, and the prediction is better for the α than for the β secondary structure. The medium- and long-range correlations seem equally well predicted for all the secondary structures. The efficiency of the method is compared to a previously proposed spectral correlation approach. Received: 5 July 2000 / Accepted: 8 September 2000 / Published online: 19 January 2001     

Exploring the use of a structural alphabet for structural prediction of protein loops

 The prediction of loop conformations is one of the challenging problems of homology modeling, owing to the large sequence variability associated with these parts of protein structures. In the present study, we introduce a search procedure that evolves in a structural alphabet space deduced from a hidden Markov model to simplify the structural information. It uses a Bayesian criterion to predict, from the amino acid sequence of a loop region, its corresponding word in the structural alphabet space. The results show that our approach ranks 30% of the target words with the best score, 50% within the five best scores. Interestingly, our approach is also suited to accept or not the prediction performed. This allows the ranking of 57% of the target words with the best score, 67% within the five best scores, accepting 16% of learned words and rejecting 93% of unknown words. Received: 17 July 2000 / Accepted: 5 January 2001 / Published online: 3 April 2001     

Atomic force microscopy study of salivary pellicles formed on enamel and glass in vivo

This study was performed to evaluate the use of atomic force microscopy (AFM) in examining the surface of the adsorbed layer of salivary proteins (salivary pellicle) formed in vivo on dental enamel and glass surfaces. Enamel and glass test pieces were attached to the buccal surfaces of the upper first molar teeth in two adults using removable intraoral splints. The splints were carried intraorally over periods ranging from 10 min to1 h. Using the contact mode of AFM, pellicle structures could be recognised on intraorally exposed specimens compared to nonexposed enamel and glass surfaces. The surface of the adsorbed salivary pellicle was characterised by a dense globular appearance. The diameter of the globulelike protein aggregates adsorbed onto enamel and glass varied between 80 and 200 nm and 80 and 150 nm, respectively. The structure of the adsorbed protein layer was clearly visible on glass surfaces, even though minor differences in the protein layer between glass and enamel specimens were observed. This study indicates that AFM is a powerful tool for high-resolution examination of the salivary pellicle surface structure in its native (hydrated) state. AFM avoids artefacts due to fixing, dehydration and sputter-coating which occur with scanning electron microscopic analyses. Received: 29 November 2000 Accepted: 14 December 2000     

Molecular dynamics simulations of the isolated domain 1 of annexin I

Annexin molecules consist of a symmetrical arrangement of four domains of identical folds but very different sequences. Nuclear magnetic resonance (NMR) experiments on the isolated domains of annexin I in aqueous solution have indicated that domain 1 retains its native structure whereas domain 2 unfolds. Therefore these two domains constitute interesting models for comparative simulations of structural stability using molecular dynamics. Here we present the preliminary results of molecular dynamics simulations of the isolated domain 1 in explicit water at 300 K, using two different simulation protocols. For the first, domain 1 was embedded in a 46 ? cubic box of water. A group-based non-bonded cut-off of 9 ? with a 5–9 ? non-bonded switching function was used and a 2 fs integration step. Bonds containing hydrogens were constrained with the SHAKE algorithm. These conditions led to unfolding of the domain within 400 ps at 300 K. In the second protocol, the domain was embedded in a 62 ? cubic box of water. An atom-based non-bonded cut-off of 8–12 ? using a force switching function for electrostatics and a shifting function for van der Waals interactions were used with a 1 fs integration step. This second protocol led to a native-like conformation of the domain in accord with the NMR data which was stable over the whole trajectory (∼2 ns). A small, but well-defined relaxation of the structure, from that observed for the same domain in the entire protein, was observed. This structural relaxation is described and methodological aspects are discussed. Received: 10 May 1998 / Accepted: 4 August 1998 / Published online: 2 November 1998     

Stabilization centers and protein stability

The well-balanced stability of protein structures allows large-scale fluctuations, which are indispensable in many biochemical functions, ensures the long-term persistence of the equilibrium structure and it regulates the degradation of proteins to provide amino acids for biosynthesis. This balance is studied in the present work with two sets of proteins by analyzing stabilization centers, defined as certain clusters of residues involved in cooperative long-range interactions. One data set contains 56 proteins, which belong to 16 families of homologous proteins, derived from organisms of various physiological temperatures. The other set is composed of 31 major histocompatibility complex (MHC)–peptide complexes, which represent peptide transporters complexed with peptide ligands that apparently contribute to the stabilization of the MHC proteins themselves. We show here that stabilization centers, which had been identified as special clusters of residues that protect the protein structure, evolved to serve also as regulators of function – related degradation of useless protein as part of protein housekeeping. Received: 25 August 2000 / Accepted: 6 September 2000 / Published online: 21 December 2000     

A statistical analytical approach to predict the secondary structure of proteins from amino acid sequence information

A statistical analytical approach has been used to analyze the secondary structure (SS) of amino acids as a function of the sequence of amino acid residues. We have used 306 non-homologous best-resolved protein structures from the Protein Data Bank for the analysis. A sequence region of 32 amino acids on either side of the residue is considered in order to calculate single amino acid propensities, di-amino acid potentials and tri-amino acid potentials. A weighted sum of predictions obtained using these properties is used to suggest a final prediction method. Our method is as good as the best-known SS prediction methods, is the simplest of all the methods, and uses no homologous sequence/family alignment data, yet gives 72% SS prediction accuracy. Since the method did not use many other factors that may increase the prediction accuracy there is scope to achieve greater accuracy using this approach. Received: 4 May 1998 / Accepted: 17 September 1998 / Published online: 10 December 1998     

Compacting local protein folds with a “hybrid protein model”

 The “hybrid protein model” is a fuzzy model for compacting local protein structures. It learns a nonredundant database encoded in a previously defined structural alphabet composed of 16 protein blocks (PBs). The hybrid protein is composed of a series of distributions of the probability of observing the PBs. The training is an iterative unsupervised process that for every fold to be learnt consists of looking for the most similar pattern present in the hybrid protein and modifying it slightly. Finally each position of the hybrid protein corresponds to a set of similar local structures. Superimposing those local structures yields an average root mean square of 3.14 ?. The significant amino acid characteristics related to the local structures are determined. The use of this model is illustrated by finding the most similar folds between two cytochromes P450. Received: 13 June 2000 / Accepted: 18 September 2000 / Published online: 19 January 2001     

Quantum mechanical/molecular mechanical study of three stationary points along the deacylation step of the catalytic mechanism of elastase

A large amount of experimental as well as theoretical information is available about the mechanism of serine proteases, but many questions remain unanswered. Here we study the deacylation step of the reaction mechanism of elastase. The water molecule in the acyl-enzyme active site, the binding mode of the carbonyl oxygen in the oxyanion hole, the characteristics of the tetrahedral intermediate structure, and the mobility of the imidazole ring of His-57 were studied with quantum mechanical/molecular mechanical methods. The models are based on a recent high-resolution crystal structure of the acyl-enzyme intermediate. The nucleophilic water in the active site of the acyl-enzyme has been shown to have two minima that differ by only 2 kcalmol⁻¹ in energy. The carbonyl group of the acyl-enzyme is located in the oxyanion hole and is positioned for attack by the hydrolytic water. The tetrahedral intermediate is a weakly bonded system, which is electrostatically stabilized by short hydrogen bonds to the backbone NH groups of Gly-193 and Ser-195 in the oxyanion hole. The short distance between the N^ɛ2 of His-57 and the O^γ of Ser-195 in the tetrahedral intermediate indicates a small movement of the imidazole ring towards the product in the deacylation step. The carbonyl group of the enzyme-product complex is not held strongly in the oxyanion hole, which shows that the peptide is first released from the oxyanion hole before it leaves the active site to regenerate the native state of the enzyme. Received: 11 September 2000 / Accepted: 15 September 2000 / Published online: 21 March 2001     

Direct and indirect band gap types in one-dimensional conjugated or stacked organic materials

Whether a semiconductor has a direct or an indirect band gap is important in determining physical properties such as photoconductivity and electroluminescence. For one-dimensional conjugated polymeric semiconductors, as well as organic molecular crystals, we show how the band gap type (direct or indirect) is determined by the shapes of the HOMO and the LUMO of constituent monomeric conjugated molecules. The connectivity of the monomer units, and the topology of orbital interaction determine the band gap type. Pairing symmetry in the π electronic system of even alternant hydrocarbons allows the immediate prediction of the band gap type, direct or indirect, by examining only the structures of the monomer units and their connectivity in polymers or molecular stacks. Received: 29 June 1998 / Accepted: 3 September 1998 / Published online: 8 February 1999     

Extracting parameters for base-pair level models of DNA from molecular dynamics simulations

 A method is described to extract a complete set of sequence-dependent energy parameters for a rigid base-pair model of DNA from molecular dynamics (MD) simulations. The method is properly consistent with equilibrium statistical mechanics and leads to effective inertia parameters for the base-pair units as well as stacking and stiffness parameters for the base-pair junctions. We give explicit formulas that yield a complete set of base-pair model parameters in terms of equilibrium averages that can be estimated from a time series generated in an MD simulation. The expressions to be averaged depend strongly both on the choice of coordinates used to describe rigid-body orientations and on the choice of strain measures at each junction. Received: 12 July 2000 / Accepted: 5 January 2001 / Published online: 3 May 2001     

A generalized Langevin dynamics approach to model solvent dynamics effects on proteins via a solvent-accessible surface. The carboxypeptidase A inhibitor protein as a model

A generalized Langevin dynamics (GLD) scheme is derived for (bio)macromolecules having internal structure, arbitrary shapes and a size larger than solvent molecules (i.e. proteins). The concept of solvent-accessible surface area (SASA) is used to incorporate solvent effects via external forces thereby avoiding its explicit molecular representation. A simulation algorithm is implemented in the GROMOS molecular dynamics (MD) program including random forces and memory effects, while solvation effects enter via derivatives of the surface area. The potato carboxypeptidase inhibitor (PCI), a small protein, is used to numerically test the approach. This molecule has N- and C-terminal tails whose structure and fluctuations are solvent dependent. A 1-ns MD trajectory was analyzed in depth. X-ray and NMR structures are used in conjunction with MD simulations with and without explicit solvent to gauge the quality of the results. All the analyses showed that the GLD simulation approached the results obtained for the MD simulation with explicit simple-point-charge-model water molecules. The SASAs of the polar atoms show a natural exposure towards the solvent direction. A FLS solvent simulation was completed in order to sense memory effects. The approach and results presented here could be of great value for developing alternatives to the use of explicit solvent molecules in the MD simulation of proteins, expanding its use and the time-scale explored. Received: 2 February 2000 / Revised: 12 March 2000 / Accepted: 26 May 2000 / Published online: 2 November 2000     

Transition structure selectivity in enzyme catalysis: a QM/MM study of chorismate mutase

Two different transition structures (TSs) have been located and characterized for the chorismate conversion to prephenate in Bacillus subtilis chorismate mutase by means of hybrid quantum-mechanical/molecular-mechanical (QM/MM) calculations. GRACE software, combined with an AM1/CHARMM24/TIP3P potential, has been used involving full gradient relaxation of the position of ca. 3300 atoms. These TSs have been connected with their respective reactants and products by the intrinsic reaction coordinate (IRC) procedure carried out in the presence of the protein environment, thus obtaining for the first time a realistic enzymatic reaction path for this reaction. Similar QM/MM computational schemes have been applied to study the chemical reaction solvated by ca. 500 water molecules. Comparison of these results together with gas phase calculations has allowed understanding of the catalytic efficiency of the protein. The enzyme stabilizes one of the TSs (TS_OHout) by means of specific hydrogen bond interactions, while the other TS (TS_OHin) is the preferred one in vacuum and in water. The enzyme TS is effectively more polarized but less dissociative than the corresponding solvent and gas phase TSs. Electrostatic stabilization and an intramolecular charge-transfer process can explain this enzymatically induced change. Our theoretical results provide new information on an important enzymatic transformation and the key factors responsible for efficient selectivity are clarified. Received: 25 March 2000 / Accepted: 7 August 2000 / Published online: 23 November 2000     

Transition state for intramolecular C–H bond cleavage in [(LCu)2(μ-O)2]2+ (L = 1,4,7-tribenzyl-1,4,7-triazacyclononane)

Hybrid quantum mechanical/molecular mechanical electronic structure calculations reveal the transition state for C–H bond cleavage in [(LCu)₂ (μ-O)₂]²⁺ (L=1,4,7-tribenzyl-1,4,7-triazacyclononane) to be consistent with a hydrogen-atom-transfer mechanism from carbon to oxygen. At the MPW1K/double-zeta effective core potential(+)|univeral force field level, 0 K activation enthalpies for the parent, p-CF3, and p-OH substituted benzyl systems are predicted to be 8.8, 9.5, and 7.8 kcal/mol. Using a one-dimensional Eckart potential to estimate quantum effects on the reaction coordinate, reaction in the unsubstituted system is predicted to proceed with a primary kinetic isotope effect of 22 at 233 K. Structural parameters associated with the hydrogen-atom transfer are consistent with the Hammond postulate. Received: 10 October 2000 / Accepted: 3 November 2000 / Published online: 3 April 2001     

Implicit two-phase solvation model as a tool to assess conformation and energetics of proteins in membrane-mimetic media

 A heterogeneous implicit membrane-mimetic model is applied to simulations of membrane proteins. The model employs atomic solvation parameters for gas–water and gas–cyclohexane transfer. It is used to analyze structure, energetics, and orientation with respect to the bilayer of two polypeptides with different modes of membrane binding – hydrophobic segment of human glycophorin A (GpA) and cytotoxin II from Naja naja oxiana snake venom (CTX). The native state of GpA represents a transmembrane (TM) α helix, while CTX is a water-soluble protein, which is able to interact with the cell membrane. The conformational space of the polypeptides was explored in Monte Carlo simulations. The results show that the most stable conformers of GpA represent a TM α helix. They are additionally stabilized by an applied TM voltage. The results also show that CTX inserts with its three loops, does not cross the hydrophobic layer, and stays partially immersed in the membrane. This agrees well with the experimental data, thus confirming the validity of the solvation model. Received: 13 June 2000 / Accepted: 15 September 2000 / Published online: 19 January 2001     

Neural networks to study invariant features of protein folding

Protein secondary structures result both from short-range and long-range interactions. Here neural networks are used to implement a procedure to detect regions of the protein backbone where local interactions have an overwhelming effect in determining the formation of stretches in α-helical conformation. Within the framework of a modular view of protein folding we have argued that these structures correspond to the initiation sites of folding. The hypothesis to be tested in this paper is that sequence identity beside ensuring similarity of the three-dimensional conformation also entails similar folding mechanisms. In particular, we compare the location and sequence variability of the initiation sites extracted from a set of proteins homologous to horse heart cytochrome c. We present evidence that the initiation sites conserve their position in the aligned sequences and exhibit a more reduced variability in the residue composition than the rest of the protein. Received: 24 April 1998 / Accepted: 4 August 1998 / Published online: 11 November 1998     

Analyzing patterns between regular secondary structures using short structural building blocks defined by a hidden Markov model

Hidden Markov models were used to identify recurrent short 3D structural building blocks (SSBBs) describing protein backbones. Polypeptide chains were broken down into successive short segments defined by their inter-alpha-carbon distances. Fitting the model to a database of nonredundant proteins identified 12 distinct SSBBs and described the preferred pathways by which SSBBs were assembled to form the 3D structure of the proteins. Protein backbones were labelled in terms of these SSBBs. The observed SSBB preferences for fragments located between regular secondary structures suggested that they depended more on the following regular structure than on the preceding one. Extraction of repeated series of SSBBs between regular secondary structures showed some structural specificity within different connection types. These results confirm that SSBBs can be used as building blocks for analyzing protein structures, and can yield new information on the structures of the coils flanking secondary structures. Received: 14 May 1998 / Accepted: 4 August 1998 / Published online: 16 November 1998     

Molecular orbital study on the OH stretching frequency of the phenol dimer and its cation

Ab initio calculations were performed to investigate the structure and bonding of the phenol dimer and its cation, especially the OH stretching frequencies. Some stable structures of the phenol dimer and its cation were obtained at the Hartree–Fock level and were found to be in agreement with predictions based on spectroscopic investigations. In these dimers the phenol moieties are bound by a single OH⋯O hydrogen bond. The hydrogen bond is much stronger in the dimer cation than in the neutral dimer. The calculated binding energy of the phenol dimer in the most stable structure was 6.5–9.9 kcal/mol at various levels of calculation, compared with the experimental value of 5 kcal/mol or greater. The binding energy of the phenol dimer cation is more than 3 times (24.1–30.6 kcal/mol) as large as that of the neutral dimer. For the phenol dimer the OH stretching frequency of the proton-accepting phenol (PAP) is 3652 cm⁻¹ and that of the proton-donating phenol (PDP) is 3516 cm⁻¹; these are in agreement with observed values of 3654 and 3530 cm⁻¹, respectively. For the phenol dimer cation the OH stretching frequency of the PAP is 3616–3618 cm⁻¹ in comparison with an observed value of 3620 ± 3 cm⁻¹. That of the PDP in the dimer cation is calculated to be 2434–2447 cm⁻¹, which is 1210–1223 cm⁻¹ lower than that of the bare phenol. The large reduction in the OH stretching frequency of the PDP in the phenol dimer cation is attributed to the formation of a stronger hydrogen bond in the cation than in the neutral dimer. Received: 24 March 2000 / Accepted: 26 April 2000 / Published online: 11 September 2000     

Analysis of the RGD sequence in protein structures: comparison to the conformations of the RGDW and DRGDW peptides determined by molecular dynamics simulations

Geometric properties of the RGD sequence in a data set of protein crystal and NMR structures deposited in the Protein Data Bank were examined to identify structural characteristics that are related to cell adhesion activity. Interatomic distances and dihedral angles are examined. These geometric measures are then used in an analysis of the conformations of the RGDW and _DRGDW peptides obtained from molecular dynamics simulations (Stote RH, et al. (2000) J Phys ChemB 104:1624). This analysis leads to the suggestion that differences in the accessible conformations contribute to the difference in biological activity between the RGDW and the _DRGDW peptides. Received: 15 August 2000 / Accepted: 4 October 2000 / Published online: 21 March 2001     

Investigations of the microscopic structure of poly(vinyl alcohol) hydrogels by confocal laser scanning microscopy

The fluorescence mode confocal laser scanning microscopy (CLSM) is introduced as an alternative method to investigate the bulk structure of poly(vinyl alcohol) (PVA) hydrogel. Investigations of the bulk structure of hydrogel samples, prepared by freezing and controlled thawing of aqueous PVA solutions followed by fluorochrome conjugation, were possible in the native state because with this technique water does not need to be removed prior to examination. This is of advantage to other methods, such as scanning electron microscopy, requiring dehydration by critical-point drying or freeze-etching, because both may result in a significant alteration of the gel structure. CLSM images of the hydrogel bulk structure were taken at several successive intervals from the surface into the hydrogel (up to 60 μm) without freeze-fracturing or cutting the sample. Detailed morphological characterization is achievable by superimposing series of images taken at successive intervals and by magnifying special regions of interest. Images of hydrogel bulk structures revealed a continuous, three-dimensional network that originates from phase-separation (spinodal decomposition) during the freezing period. The pore or mesh size in the cryogel increased, from about 2–7 μm, with decreasing PVA concentration. The surface layer was only a few microns thick, and the bulk structure underneath showed neither porosity gradients nor structural orientations. Received: 29 April 2000/Accepted: 18 August 2000     

Prediction of solvent accessibility of amino acid residues: critical aspects

For predicting solvent accessibility from the sequence of amino acids in proteins, we use a logistic function trained on a non-redundant protein database. Using a principal component analysis, we find that the prediction can be considered, in a good approximation, as a monofactorial problem: a crossed effect of the burial propensity of amino acids and of their locations at positions flanking the amino acid of interest. Complementary effects depend on the presence of certain amino acids (mostly P, G and C) at given positions. We have refined the predictive model (1) by adding supplementary input data, (2) by using a strategy of prediction correction and (3) by adapting the decision rules according to the amino acid type. We obtain a best score of 77.6% correct prediction for a relative accessibility of 9%. However, compared to trivial strategy only based upon the frequencies of buried or exposed residues, the gain is less than 4%. Received: 4 June 1998 / Accepted: 17 September 1998 / Published online: 10 December 1998