Stabilization centers in various proteins

Interactions among residues together with their interactions with the surrounding medium determine the unique structure of globular proteins. An algorithm was recently developed to locate residues participating in cooperative long-range interactions, called stabilization center residues, that are primarily responsible for preventing the decay of the 3D structure. While our statistical analysis showed that interactions of stabilization center residues hardly influence the formation of the various secondary structure elements, the distribution of the stabilization center residues is rather uneven among the secondary structure elements. Here we analyzed the frequency and distribution of the stabilization center residues and their interacting pairs in secondary structure classes to learn about the effect of secondary structure on the formation and properties of stabilization centers and about the types of interactions responsible for stabilization of proteins of various secondary structure classes. It was found that residues from the same secondary structure tend to interact with each other in the stabilization centers of all classes. It is also suggested that the folding-unfolding equilibrium is governed by different principles for class all-α than for the rest of the classes. Received: 24 April 1998 / Accepted: 17 September 1998 / Published online: 7 December 1998     

Assessment of conformation and energetics of the N-terminal part of elafin via computer simulations

Elafin, a specific inhibitor of elastase, is thought to play a regulatory role in inflammation. An NMR-derived solution structure of recombinant elafin has been reported [Francart et al. (1997) J Mol Biol 268:666 ], although the conformation of its flexible N-terminal part is not established. There is experimental evidence that the N terminus (residues 1–15) of elafin interacts with the cell membrane. To explore the conformational preferences of residues in this region, we have performed Monte Carlo simulations of the peptide in water, in cyclohexane, and in a model membrane. Additionally, 3.7-ns molecular dynamics with explicit water was carried out. The main results were that the hydrophobic environment stabilizes an α helix in the region 6–11, the peptide is unordered in water, and it is attached to the membrane via the amphiphilic α-helix 6–11, which inserts with its N terminus forming an angle of about 60° to the membrane plane. We therefore assume that in nonpolar media the N-terminal part of elafin forms a short α helix which might act as a membrane anchor. Received: 5 July 2000 / Accepted: 4 October 2000 / Published online: 28 February 2001     

Ab initio structure predictions using a hierarchical approach applied to 434 cro and the Drosophila homeodomain

A discrete-state ab initio protein structure prediction procedure is presented, based on the assumption that some proteins fold in an hierarchical way, where the early folding of independent units precedes and helps complete structure formation. It involves a first step predicting, by means of threading algorithms and local structure prediction methods, the location of autonomous protein subunits presenting favorable local and tertiary interactions. The second step consists of predicting the structure of these units by Monte Carlo simulated annealing using several database-derived potentials. In a last step, these predicted structures are used as starting conformations of additional simulations, keeping these structures frozen and including the complete protein sequence. This procedure is applied to two small DNA-binding proteins, 434 cro and the Drosophila melanogaster homeodomain that contain 65 and 47 residues, respectively, and is compared to the nonhierarchical procedure where the whole protein is predicted in a single run. The best predicted structures were found to present root-mean-square deviations relative to the native conformation of 2.7 ? in the case of the homeodomain and of 3.9 ? for 434 cro; these structures thus represent low-resolution models of the native structures. Strikingly, not only the helices were correctly predicted but also intervening turn motifs. Received: 6 July 2000 / Accepted: 8 September 2000 / Published online: 21 December 2000     

Influence of ligand binding on the conformation of Torpedo californica acetylcholinesterase

With the aim of identifying structural changes in acetylcholinesterase, induced by ligand binding, we use a completely automatic procedure to analyse the differences between the backbone conformation of the free enzyme and those in eight complexes of Torpedo californica acetylcholinesterase, with various quaternary ammonium ligands, and with the protein inhibitor fasciculin. In order to discriminate between structural changes due to ligand binding and those arising from model imprecision, we also examine protein–ligand and protein–water contacts. Except for the peptide flip in the complex with huperzine A, the backbones of other complexes with quaternary ammonium ligands display negligible changes relative to the free enzyme. Another exception is the complex with the bisquaternary ammonium ligand decamethonium, where several loops display above average deformations, but only two, those spanning residues 334–348 and residues 277–304, seem to move as a result of ligand binding. Movement of the ω loop (residues 61–95) is detected only in the complex with the protein fasciculin. Received: 21 July 2000 / Accepted: 18 September 2000 / Published online: 28 February 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     

Deciphering globular protein sequence–structure relationships: from observation to prediction

Careful comparison of proteins sharing a same fold but only low or no sequence identity should allow a better understanding of the coding of three-dimensional structures by amino acid sequences. It has already been shown that positions of a given fold occupied mainly by hydrophobic residues in the different proteins of a structural family share very specific physical properties and participate in stabilization of the protein domain. They probably also play a crucial role in the very first steps of folding [ Poupon A, Mornon J.-P (1999) FEBS Lett. 452: 283–289; Mirny LA, Shaknovich EI (1999) J. Mol. Biol. 291: 177–196]. To further understand the sequence–structure relationship, we studied the correlation between allowed mutations at a given three-dimensional position and some of its physical properties. The different amino acids were divided in three groups (hydrophobic, nonpolar or weakly polar and polar or charged), and a correlation was established between the occupation rate of each group at a given position in the fold and the burying, the side-chain dispersion, the interposition distances and the ability to form a network of directly interacting residues. The results are then applied to predict some solvent accessibility. We show that this property can be accurately predicted for about 70% of the residues, providing precious information concerning the corresponding three-dimensional structures. The results are used to predict other structural features, as secondary structures, compactness or long-range interactions between residues remote in sequence. This information will allow the number of possible structures for a given sequence to be reduced considerably, simplifying the ab initio modelling problem to a level where it might be solved by computing methods. Received: 7 October 2000 / Accepted: 5 January 2001 / Published online: 3 April 2001     

Information about the biologically relevant properties of Clostridium pasteurianum rubredoxin obtained from modeling and dynamics simulations of molecular variants

Rubredoxins are small electron transfer proteins containing one iron atom at their active site. The rubredoxin from the anaerobic bacterium Clostridium pasteurianum has been subjected to molecular dynamics studies starting from the minimized solvated structure. The results of the simulations have been compared with identical ones carried out with selected mutated forms of the protein obtained by molecular modeling. Surface residues, which are highly conserved among rubredoxins and close to the cysteine ligands, can be replaced by glutamates, i.e. long chain carboxylates. The main structural consequence is a shift of the protein backbone bearing conserved aromatic residues. Reciprocally, substitution of the aromatic residue closest to the iron atom shifts the cysteine-containing peptide fragments. These observations have been related to the changes in electron transfer and redox properties previously measured for this set of rubredoxin molecular variants. Received: 16 May 1998 / Accepted: 4 August 1998 / Published online: 2 November 1998     

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     

Prediction of stability changes upon single-site mutations using database-derived potentials

One of the purposes of studying protein stability changes upon mutations is to get information about the dominating interactions that drive folding and stabilise the native structure. With this in mind, we present a method that predicts folding free-energy variations caused by point mutations using combinations of two types of database-derived potentials, i.e. backbone torsion-angle potentials and distance potentials, describing local and non-local interactions along the chain, respectively. The method is applied to evaluate the folding free-energy changes of 344 single-site mutations introduced in six different proteins and a synthetic peptide. We found that the relative importance of local versus non-local interactions along the chain is essentially a function of the solvent accessibility of the mutated residues. For the subset of totally buried residues, the optimal potential is the sum of a distance potential and a torsion potential weighted by a factor of 0.4. This combination yields a correlation coefficient between measured and computed changes in folding free energy of 0.80. For mutations of partially buried residues, the best potential is the sum of a torsion potential and a distance potential weighted by 0.7. For fully accessible residues, the torsion potentials taken alone perform best, reaching correlation coefficients of 0.87 on all but 10 mutations; the excluded mutations seem to modify the backbone structure or to involve interactions that are atypical for the surface. These results show that the relative weight of non-local interactions along the sequence decreases as the solvent accessibility of the mutated residue increases, and vanishes at the protein surface. On the contrary, the weight of local interactions increases with solvent accessibility. The latter interactions are nevertheless never negligible, even for the most buried residues. Received: 20 May 1998 / Accepted: 3 September 1998 / Published online: 7 December 1998     

Coupling overall rotations with modal dynamics

 We consider a model for protein dynamics in which only certain collective, global motions are allowed. These directions are given by the slowest harmonics modes, as given in the reference frame of the protein. Furthermore, the latter is allowed to rotate and translate in response to interactions with other molecules. The model is obtained by projecting the (averaged) Newton equations onto this set of harmonic modes. We show that the subsequent homogenization of the time scales allows time steps one order of magnitude larger than the standard ones. This homogenization is also shown to be a necessary ingredient in order to get meaningful statistics of the trajectory. Received: 3 July 2000 / Accepted: 15 September 2000 / Published online: 21 December 2000     

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     

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     

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 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     

Fragmentation methods on the balance: unambiguous top–down mass spectrometric characterization of oxaliplatin–ubiquitin binding sites

The interaction between oxaliplatin and the model protein ubiquitin (Ub) was investigated in a top–down approach by means of high-resolution electrospray ionization mass spectrometry (ESI-MS) using diverse tandem mass spectrometric (MS/MS) techniques, including collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD), and electron transfer dissociation (ETD). To the best of our knowledge, this is the first time that metallodrug–protein adducts were analyzed for the metal-binding site by ETD-MS/MS, which outperformed both CID and HCD in terms of number of identified metallated peptide fragments in the mass spectra and the localization of the binding sites. Only ETD allowed the simultaneous and exact determination of Met1 and His68 residues as binding partners for oxaliplatin. CID-MS/MS experiments were carried out on orbitrap and ion cyclotron resonance (ICR)-FT mass spectrometers and both instruments yielded similar results with respect to number of metallated fragments and the localization of the binding sites. A comparison of the protein secondary structure with the intensities of peptide fragments generated by collisional activation of the [Ub + Pt-(chxn)] adduct [chxn = (1R,2R)-cyclohexanediamine] revealed a correlation with cleavages in solution phase random coil areas, indicating that the N-terminal β-hairpin and α-helix structures are retained in the gas phase.     

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     

A new “hydrophobic template” method detects segments forming transmembrane α-helical bundles in ion channels

We present a “hydrophobic template” method enabling recognition of α-helix bundles in membrane channels from sequence analysis. Inspection of hydrophobic properties of pore-forming helices in proteins with known structure (A-B₅ toxins) permits delineation of a common polarity motif: two hydrophobic surface stretches separated by polar areas. The bundles are stabilized by nonpolar interhelical contacts. A number of transmembrane segments were checked for presence of this motif, and it was detected for pore-forming helices of several ion transporters (segments M2 of acetylcholine and GABA_A receptors, α5 peptide of δ-endotoxin), which reveal five α-helix bundle architecture. Applications of the method to modeling of membrane channels are discussed. Received: 24 April 1998 / Accepted: 3 September 1998 / Published online: 7 December 1998     

Prediction of protein structure using a knowledge-based off-lattice united-residue force field and global optimization methods

A united-residue model of polypeptide chains developed in our laboratories with united side-chains and united peptide groups as interaction sites is presented. The model is designed to work in continuous space; hence efficient global-optimization methods can be applied. In this work, we adopted the distance-scaling method that is based on continuous deformation of the original rugged energy hypersurface to obtain a smoothed surface. The method has been applied successfully to predict the structures of simple motifs, such as the three-helix bundle structure of the 10-58 fragment of staphylococcal protein A in de novo folding simulations and more complicated motifs in inverse-folding simulations. Received: 24 April 1998 / Accepted: 4 August 1998 / Published online: 2 November 1998     

Analysis of nitrated proteins and tryptic peptides by HPLC-chip-MS/MS: site-specific quantification,nitration degree,and reactivity of tyrosine residues

The reaction products and pathways of protein nitration were studied with bovine serum albumin (BSA) and ovalbumin (OVA) nitrated by liquid tetranitromethane (TNM) or by gaseous nitrogen dioxide and ozone (NO₂ + O₃). Native and nitrated proteins were enzymatically digested with trypsin, and the tryptic peptides were analyzed by high-performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) using a chip cube nano-flow system (Agilent). Upon nitration by TNM, up to ten of 17 tyrosine residues in BSA and up to five of ten tyrosine residues in OVA could be detected in nitrated form. Upon nitration by NO₂ + O₃, only three nitrated tyrosine residues were found in BSA. The nitration degrees of individual nitrotyrosine residues (ND_Y) were determined by site-specific quantification and compared to the total protein nitration degrees (ND) determined by photometric detection of HPLC-DAD. The slopes of the observed linear correlations between ND_Y and ND varied in the range of ~0.02–2.4 for BSA and ~0.2–1.6 for OVA. They provide information about the relative rates of nitration or reaction probabilities for different tyrosine residues. In BSA, the tyrosine residue Y₁₆₁ was by far most reactive against NO₂ + O₃ and one of the four most reactive positions with regard to nitration by TNM. In OVA, all except one tyrosine residue detected in nitrated form exhibited similar reactivities. The observed nitration patterns show how the site selectivity of protein nitration depends on the nitrating agent, reaction conditions, and molecular structure of the protein (primary, secondary, and tertiary).     

Linear scaling computation of the Fock matrix. III. Formation of the exchange matrix with permutational symmetry

 A direct comparison is made between two recently proposed methods for linear scaling computation of the Hartree–Fock exchange matrix to investigate the importance of exploiting two-electron integral permutational symmetry. Calculations on three-dimensional water clusters and graphitic sheets with different basis sets and levels of accuracy are presented to identify specific cases where permutational symmetry may or may not be useful. We conclude that a reduction in integrals via permutational symmetry does not necessarily translate into a reduction in computation times. For large insulating systems and weakly contracted basis sets the advantage of permutational symmetry is found to be negligible, while for noninsulating systems and highly contracted basis sets a fourfold speedup is approached. Received: 8 October 1999 / Accepted: 3 January 2000 / Published online: 21 June 2000