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Biomedical subjects

J Moult

Publications and source records attributed to J Moult.

At least 37 records · Page 2Linked to original sources
PubMed1997

Ab initio protein folding simulations with genetic algorithms: simulations on the complete sequence of small proteins.

Ab-initio folding simulations have been performed on three small proteins using a genetic algorithm- (GA-) based search method which operates on an all atom representation. Simulations were also performed on a number of small peptides expected to be independent folding units. The present genetic algorithm incorporates the results of developments made to the method first tested in CASP1. Additional operators have been introduced into the search in order to allow the simulation of longer sequences and to avoid the simulation of longer sequences and to avoid premature free energy convergence. Secondary structure information derived from a consensus of eight methods and Monte Carlo simulations on sets of homologous sequences has been used to bias the starting populations used in the GA simulations. For the fragment simulations, the results generally have approximately correct local structure, but tend to be too compact, leading to poor RMS error values. One of the three small protein structures has the topology and most of the general organization correct, although many of the details are incorrect.

Algorithms↗
PubMed1997

Handling context-sensitivity in protein structures using graph theory: bona fide prediction.

We constructed five comparative models in a blind manner for the second meeting on the Critical Assessment of protein Structure Prediction methods (CASP2). The method used is based on a novel graph-theoretic clique-finding approach, and attempts to address the problem of interconnected structural changes in the comparative modeling of protein structures. We discuss briefly how the method is used for protein structure prediction, and detail how it performs in the blind tests. We find that compared to CASP1, significant improvements in building insertions and deletions and sidechain conformations have been achieved.

Amino Acid Sequence↗
PubMed1997

Criteria for evaluating protein structures derived from comparative modeling.

Following the first experiment for the Critical Assessment of methods for protein Structure Prediction (CASP1), numerical criteria were devised to analyze the performance of prediction methods. We report here the criteria for comparative modeling, and how effective they were in CASP2. These criteria are intended to evolve into a set of numerical measures that provide a comprehensive assessment of the quality of a structure produced by comparative modeling, and provide a means of investigating which modeling methods are most effective, so as to establish where future effort may be most productively applied.

Evaluation Studies as Topic↗
PubMed1997

Comparison of database potentials and molecular mechanics force fields.

The advantages and disadvantages of database and molecular mechanics force fields for the study of macromolecules are compared, with emphasis on the ability to distinguish between correct and incorrect structures. Molecular mechanics force fields have the advantage of resting on a clear theoretical basis, permitting an in-depth analysis of different contributions. On the other hand, large simplifications are necessary for tractable computing, and there has so far been little effective testing at the macromolecular level. Database potentials allow greater freedom of functional form and have been shown to be effective at discriminating between correct and incorrect complete structures. The principal negative is a controversial relationship to free energy. More testing and comparison of both sorts of potential are needed.

Databases, Factual↗
PubMed1997

Chaos in protein dynamics.

MD simulations, currently the most detailed description of the dynamic evolution of proteins, are based on the repeated solution of a set of differential equations implementing Newton's second law. Many such systems are known to exhibit chaotic behavior, i.e., very small changes in initial conditions are amplified exponentially and lead to vastly different, inherently unpredictable behavior. We have investigated the response of a protein fragment in an explicit solvent environment to very small perturbations of the atomic positions (10(-3)-10(-9) A). Independent of the starting conformation (native-like, compact, extended), perturbed dynamics trajectories deviated rapidly, leading to conformations that differ by approximately 1 A RMSD within 1-2 ps. Furthermore, introducing the perturbation more than 1-2 ps before a significant conformational transition leads to a loss of the transition in the perturbed trajectories. We present evidence that the observed chaotic behavior reflects physical properties of the system rather than numerical instabilities of the calculation and discuss the implications for models of protein folding and the use of MD as a tool to analyze protein folding pathways.

Bacterial Proteins↗
PubMed1996

Elimination of the hydrolytic water molecule in a class A beta-lactamase mutant: crystal structure and kinetics.

Two site-directed mutant enzymes of the class A beta-lactamase from Staphylococcus aureus PC1 were produced with the goal of blocking the site that in the native enzyme is occupied by the proposed hydrolytic water molecule. The crystal structures of these two mutant enzymes, N170Q and N170M, have been determined and refined at 2.2 and 2.0 A, respectively. They reveal that the side chain of Gln 170 displaces the water molecule, whereas that of Met170 does not. In both cases, the catalytic rates with benzylpenicillin are reduced by 10(4) compared with the native enzyme. With nitrocefin, the N170Q mutant enzyme exhibits an approximately 800-fold reduced rate compared with the native enzyme and in addition, a fast initial burst with stoichiometry of 1 mol of degraded nitrocefin/mol of enzyme. Stopped-flow kinetic experiments establish that the rate constant of the burst is 250 s-1, a value comparable with the rate of acylation of the native enzyme. Two structurally based mechanisms that explain the kinetic properties of the N170Q beta-lactamase are proposed, both invoking a deacylation-impaired enzyme due to the elimination of the hydrolytic water molecule. The catalytic rate of the N170M mutant enzyme with nitrocefin is reduced by approximately 50-fold compared with the native enzyme, and the slow progressive inhibition that is revealed indicates that the hydrolysis proceeds via a branched pathway mechanism. This is consistent with the structural data that show that the water site is preserved and that Met170 occupies part of the space that is required for substrate binding. The short contacts between the substrate and the enzyme may lead to structure perturbation and inactivation.

Base Sequence↗
PubMed1996

Local interactions dominate folding in a simple protein model.

Recent computational studies of simple models of protein folding have concluded that a pronounced energy minimum (i.e. large gap in energy between low-energy states of the model) is a necessary and sufficient condition to ensure folding of a sequence to its lowest-energy conformation. Here, we show that this conclusion strongly depends on the particular temperature scheme selected to govern the simulations. On the other hand, we show that there is a dominant factor determining if a sequence is foldable. That is, the strength of possible interactions between residues close in the sequence. We show that sequences with many possible strong local interactions (either favorable or, more surprisingly, a mixture of strong favorable and unfavorable ones) are easy to fold. Progressively increasing the strength of such local interactions makes sequences easier and easier to fold. These results support the idea that initial formation of local substructures is important to the foldability of real proteins.

Mathematical Computing↗
PubMed1996

Positive and negative cooperativities at subsequent steps of oxygenation regulate the allosteric behavior of multistate sebacylhemoglobin.

Cross-linked human hemoglobin (HbA) is obtained by reaction with bis(3,5-dibromosalicyl) sebacate. Peptide maps and crystallographic analyses confirm the presence of the 10 carbon atom long sebacyl residue cross-linking the two beta82 lysines of the beta-cleft (DecHb). The Adair's constants, obtained from the oxygen binding isotherms, show that at the first step of oxygenation normal hemoglobin and DecHb have a very similar oxygen affinity. In DecHb negative binding cooperativity is present at the second step of oxygenation, which has an affinity 27 times lower than at the first step. Positive cooperativity is present at the third binding step, whose affinity is 380 times that of the second step. The fourth binding step shows a weak negative cooperativity with an affinity one-half that of the third step. Crystals of deoxy-DecHb diffracted to 1.9 angstroms resolution. The resulting atomic coordinates are very similar to those of Fermi et al. [(1984) J. Mol.Biol. 175, 159-174] and Fronticelli et al. [(1994) J. Biol Chem. 269, 23965-23969] for deoxy-HbA. The electron density map of deoxy-DecHb indicates the presence of the 10 carbon bridge between the beta82 lysines. Molecular modeling confirms that insertion of the linker into the T structure requires only slight displacement of the two beta82 lysines. Instead, insertion of the linker into the R and R2 structures [Shaanan (1983) J. Mol. Biol. 171, 31-59; Silva et al. (1992) J. Biol. Chem. 267, 17248-17256] is hindered by serious sterical restrictions. The linker primarily affects the partially and fully liganded states of hemoglobin. The data suggest in DecHb concerted conformational changes at each step of oxygenation.

Allosteric Regulation↗
PubMed1996

Crystallographic, molecular modeling, and biophysical characterization of the valine beta 67 (E11)-->threonine variant of hemoglobin.

The crystal structure of the mutant deoxyhemoglobin in which the beta-globin Val67(E11) has been replaced with threonine [Fronticelli et al. (1993) Biochemistry 32, 1235-1242] has been determined at 2.2 A resolution. Prior to the crystal structure determination, molecular modeling indicated that the Thr67(E11) side chain hydroxyl group in the distal beta-heme pocket forms a hydrogen bond with the backbone carbonyl of His63(E7) and is within hydrogen-bonding distance of the N delta of His63(E7). The mutant crystal structure indicates only small changes in conformation in the vicinity of the E11 mutation confirming the molecular modeling predictions. Comparison of the structures of the mutant beta-subunits and recombinant porcine myoglobin with the identical mutation [Cameron et al. (1993) Biochemistry 32, 13061-13070] indicates similar conformations of residues in the distal heme pocket, but there is no water molecule associated with either of the threonines of the beta-subunits. The introduction of threonine into the distal heme pocket, despite having only small perturbations in the local structure, has a marked affect on the interaction with ligands. In the oxy derivative there is a 2-fold decrease in O2 affinity [Fronticelli et al. (1993) Biochemistry 32, 1235-1242], and the rate of autoxidation is increased by 2 orders of magnitude. In the CO derivative the IR spectrum shows modifications with respect to that of normal human hemoglobin, suggesting the presence of multiple CO conformers. In the nitrosyl derivative an interaction with the O gamma atom of Thr67(E11) is probably responsible for the 10-fold increase in the rate of NO release from the beta-subunits. In the aquomet derivative there is a 6-fold decrease in the rate of hemin dissociation suggesting an interaction of the Fe-coordinated water with the O gamma of Thr67(E11).

Biophysical Phenomena↗
PubMed1996

The current state of the art in protein structure prediction.

The capabilities of current protein structure prediction methods have been assessed from the outcome of a set of blind tests. In comparative modeling, many of the numerical methods did not perform as well as expected, although the resulting structures are still of great practical use. The new methods of fold identification ('threading') were partially successful, and show considerable promise for the future. Except for secondary structure data, results from traditional ab initio methods were poor. A second blind prediction experiment is underway, and progress in all areas is expected.

Forecasting↗
PubMed1996

Genetic algorithms for protein structure prediction.

Genetic algorithms are a general class of search methods that mimic natural gene-based optimization mechanisms. Mutation, cross-over and replication operations are performed on strings. When applied to structure prediction, each string describes a particular conformation of a protein molecule. There are many ways in which such search methods may be implemented. Recent results show potential for helping with protein structure prediction, but more data are needed before a complete assessment can be made.

Algorithms↗
PubMed1996

The acute rise in plasma fibrinogen concentration with exercise is influenced by the G-453-A polymorphism of the beta-fibrinogen gene.

We have investigated the effects of chronic physical training and acute intensive exercise on plasma fibrinogen levels and the relationship of these responses to beta-fibrinogen G-453-A polymorphism genotype. One hundred fifty-six male British Army recruits were studied at the start of their 10-week basic training, which emphasizes physical fitness. Cohorts were restudied between 0.5 and 5 days after a major 2-day strenuous military exercise (ME) undertaken in their final week of training. Changes in fibrinogen concentration were adjusted for the effects of age, body mass index, and smoking history. Compared with baseline values, fibrinogen concentrations were significantly lower (11.9%, P=.04) at day 5 after ME, consistent with the beneficial effect of training. However, they were higher on days 1 through 3 after ME (suggesting an "acute-phase" response to strenuous exercise) and were maximal on days 1 and 2 (27.2%, P<.001 and 37.1%, P<.001 respectively). Fibrinogen genotype was available in 149 individuals. As expected from previous studies, men with one or more fibrinogen gene A-453 alleles had plasma fibrinogen concentration slightly but significantly higher at baseline (4.5%, P=.11). During the acute-phase response (days 2 and 3), however, the degree of rise was strongly related to the presence of the A allele, being 26.7+/-5.4% (mean+/-SE), 36.5+/-11.0%, and 89.2+/-30.7 for the GG, GA, and AA genotypes, respectively (P=.01). These results confirm that chronic exercise training lowers plasma fibrinogen levels, that intensive exercise generates an acute-phase rise in levels, and that this acute response is strongly influenced by the G/A polymorphism of the beta-fibrinogen gene.

Adolescent↗
PubMed1995

Structure, dynamics and energetics of initiation sites in protein folding: I. Analysis of a 1 ns molecular dynamics trajectory of an early folding unit in water: the helix I/loop I-fragment of barnase.

The dynamic and energetic behavior of an initiation site of protein folding (helix I/loop I fragment of barnase) isolated from the tertiary environment of the rest protein is investigated in a 1 ns molecular dynamics simulation. All atom representation, explicit solvent description, and periodic boundary conditions are applied. In the course of the simulation several steps of structural disintegration are observed, followed by events partially rebuilding the initial structure. The phase of disintegration results in a fragment conformation completely lacking hydrogen bonds, with one residue in the center of the helix changed from alpha to beta conformation. The transition state of helix disintegration is characterized by a complete i-->i + 4/i + 5 hydrogen bonding network which undergoes gradual hydrolysis starting at the solvent exposed flank and proceeding towards the interior of the fragment perpendicular to the axis of the helix. Energetic analysis of the helix transitions shows that the i-->i + 4/i-->i + 5 network of hydrogen bonds accommodates one helical residue in beta conformation with only slightly worse hydrogen bonding energy and Van der Waals packing compared to the regular alpha-helix. The stability of the fragment is primarily due to hydrophobic interactions of residues shown to be essential in mutagenesis experiments.

Amino Acid Sequence↗
PubMed1995

Role of electrostatic screening in determining protein main chain conformational preferences.

Amino acids display significant variation in propensity for the alpha R-helical, beta-sheet, and other main chain conformational states in proteins and peptides. The physical reason for these preferences remains controversial. Conformational entropy, steric factors, and the hydrophobic effect have all been advanced as the dominant underlying cause. In this work, we explore the role of a fourth factor, electrostatics, in determining the main chain conformation in protein molecules. Potentials of mean force derived from experimental protein structures are used to evaluate the free energy of electrostatic and other interactions of a residue in a protein environment. The local and nonlocal electrostatic interactions of main chain polar atoms are found to be crucial for determining the preferences of residues for the alpha R-helical state and other main chain conformational states of a residue. Further, the strength of local and nonlocal electrostatic interactions is shown to depend on the electrostatic screening by solvent and protein groups. Residue specific modulation of this screening in a manner related to side chain bulk and squatness produces a model that fits the observed distribution of residue conformations in proteins and recent experimental mutagenesis data on protein stability better than any other single factor.

Electrochemistry↗
PubMed1995

Determination of the conformation of folding initiation sites in proteins by computer simulation.

Experimental evidence and theoretical models both suggest that protein folding begins by specific short regions of the polypeptide chain intermittently assuming conformations close to their final ones. The independent folding properties and small size of these folding initiation sites make them suitable subjects for computational methods aimed at deriving structure from sequence. We have used a torsion space Monte Carlo procedure together with an all-atom free energy function to investigate the folding of a set of such sites. The free energy function is derived by a potential of mean force analysis of experimental protein structures. The most important contributions to the total free energy are the local main chain electrostatics, main chain hydrogen bonds, and the burial of nonpolar area. Six proposed independent folding units and four control peptides 11-14 residues long have been investigated. Thirty Monte Carlo simulations were performed on each peptide, starting from different random conformations. Five of the six folding units adopted conformations close to the experimental ones in some of the runs. None of the controls did so, as expected. The generated conformations which are close to the experimental ones have among the lowest free energies encountered, although some less native like low free energy conformations were also found. The effectiveness of the method on these peptides, which have a wide variety of experimental conformations, is encouraging in two ways: First, it provides independent evidence that these regions of the sequences are able to adopt native like conformations early in folding, and therefore are most probably key components of the folding pathways. Second, it demonstrates that available simulation methods and free energy functions are able to produce reasonably accurate structures. Extensions of the methods to the folding of larger portions of proteins are suggested.

Algorithms↗
PubMed1995

Confronting the problem of interconnected structural changes in the comparative modeling of proteins.

Comparative models of three proteins have been built using a variety of computational methods, heavily supplemented by visual inspection. We consider the accuracy obtained to be worse than expected. A careful analysis of the models shows that a major reason for the poor results is the interconnectedness of the structural differences between the target proteins and the template structures they were modeled from. Side chain conformations are often determined by details of the structure remote in the sequence, and can be influenced by relatively small main chain changes. Almost all of the regions of substantial main chain conformational change interact with at least one other such region, so that they often cannot be modeled independently. Visual inspection is sometimes effective in correcting errors in sequence alignment and in spotting when an alternative template structure is more appropriate. We expect some improvements in the near future through the development of structure-based sequence alignment tools, side chain interconnectedness rotamer choice algorithms, and a better understanding of the context sensitivity of conformational features.

Amino Acid Sequence↗