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MOTIVATION: The sizes of protein domains observed in the 3D-structure database follow a surprisingly narrow distribution. Structural domains are furthermore formed from a single-chain continuous segment in over 80% of instances. These observations imply that some choices of domain boundaries on an otherwise uncharacterized sequence are more likely than others, based solely on the size and segment number of predicted domains. This property might be used to guess the locations of protein domain boundaries. RESULTS: To test this possibility we enumerate putative domain boundaries and calculate their relative likelihood under a probability model that considers only the size and segment number of predicted domains. We ask, in a cross-validated test using sequences with known 3D structure, whether the most likely guesses agree with the observed domain structure. We find that domain boundary predictions are surprisingly successful for sequences up to 400 residues long and that guessing domain boundaries in this way can improve the sensitivity of threading analysis.

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Using a benchmark set of structurally similar proteins, we conduct a series of threading experiments intended to identify a scoring function with an optimal combination of contact-potential and sequence-profile terms. The benchmark set is selected to include many medium-difficulty fold recognition targets, where sequence similarity is undetectable by BLAST but structural similarity is extensive. The contact potential is based on the log-odds of non-local contacts involving different amino acid pairs, in native as opposed to randomly compacted structures. The sequence profile term is that used in PSI-BLAST. We find that combination of these terms significantly improves the success rate of fold recognition over use of either term alone, with respect to both recognition sensitivity and the accuracy of threading models. Improvement is greatest for targets between 10 % and 20 % sequence identity and 60 % to 80 % superimposable residues, where the number of models crossing critical accuracy and significance thresholds more than doubles. We suggest that these improvements account for the successful performance of the combined scoring function at CASP3. We discuss possible explanations as to why sequence-profile and contact-potential terms appear complementary.

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Tocainide is effective in the symptomatic treatment of myotonic syndromes for its ability to reduce the high frequency discharges of action potentials typical of the disease, by blocking voltage-gated sodium channels. However, its use is restricted by serious side effects. In spite of its chiral structure, tocainide is clinically used as a racemic mixture. Since the optical isomers may differ in their efficacy and toxicity, the present study was aimed at evaluating the antimyotonic activity of the pure R(-) and S(+) enantiomers of tocainide, on the abnormal membrane hyperexcitability of external intercostal muscle fibers of congenitally myotonic goats. The excitability parameters were recorded in vitro by means of the standard two-microelectrode current-clamp technique before and after the addition of the compounds. The R(-) enantiomer of tocainide at concentrations as low as 10 microM potently counteracted the abnormal excitability of myotonic fibers, by increasing the threshold current, and decreasing the latency of the action potential and firing capability. Also, this concentration of R-(-) tocainide almost completely abolished the abnormal spontaneous electrical activity occurring in about 70-80% of the myotonic fiber. The S(+) enantiomer was remarkably less potent since up to 100 microM did not restore the normal excitability pattern. The results show that most of the antimyotonic activity of tocainide resides in the R(-) enantiomer suggesting that its clinical use may allow a significant reduction of the doses and possibly of the side effects.

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Three-dimensional structures are now known for roughly half of all protein families. It is thus quite likely, in searching sequence databases, that one will encounter a homolog with known structure and be able to use this information to infer structure-function properties. The goal of Entrez's 3D structure database is to make this information accessible and useful to molecular biologists. To this end, Entrez's search engine provides three powerful features: (i) Links between databases; one may search by term matching in Medline((R)), for example, and link to 3D structures reported in these articles. (ii) Sequence and structure neighbors; one may select all sequences similar to one of interest, for example, and link to any known 3D structures. (iii) Sequence and structure visualization; identifying a homolog with known structure, one may view a combined molecular-graphic and alignment display, to infer approximate 3D structure. Entrez's MMDB (Molecular Modeling DataBase) may be accessed at: http://www.ncbi.nlm.nih.gov/Entrez/structure.html

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We have attempted to predict the three-dimensional structures of 19 proteins for the CASP3 experiment, each showing less than 25% sequence identity with known structures. Predictions were based on a threading method that aligns the target sequence with the conserved cores of structural templates, as identified from structure-structure alignments of the template with homologous neighbors. Alternative alignments were scored using contact potentials and a position-specific score matrix derived from sequence neighbors of the template. We find that this method identified the correct structural family for 11 of the 19 targets and predicted the remaining 8 targets to be similar to "none" of the templates, avoiding false positives. Threading alignments are relatively accurate for 10 of the 11 targets, including alignments for 6 of 7 identified at CASP3 as fold-recognition targets. These predictions were ranked "first place" by the CASP3 assessor when compared to fold-recognition predictions made by other methods. It appears that threading with family-specific models for structure and sequence conservation has improved threading prediction accuracy.

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We present a retrospective analysis of CASP3 threading predictions, applying evaluation and assessment criteria used at CASP2. Our purpose is twofold. First, we wish to ask whether measures of model accuracy are comparable between CASP3 and CASP2, even though they have been calculated differently. We find that these quantities are effectively the same, and that either may be used to compare model accuracy. Secondly, we wish to assess progress in fold recognition by comparing the numbers of CASP2 and CASP3 models that cross specific accuracy thresholds. We find that the number of accurate models at CASP3 drops sharply as the targets become more difficult, with less extensive similarity to known structures, exactly the pattern seen at CASP2. CASP3 teams do not seem to have predicted accurate models for targets of greater difficulty, and for a given difficulty range the best CASP3 models seem no more accurate than the best models at CASP2. At CASP3, however, we find greater numbers of accurate models for medium-difficulty targets, with extensive similarity to a known structure but no shared sequence motifs. Threading methods would appear to have become more reliable for modeling based on remote evolutionary relationships.

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1. Using whole-cell patch-clamping and Sf-9 cells expressing the rat skeletal muscle chloride channel, rCIC-1, the cellular mechanism responsible for the myotonic side effects of clofibrate derivatives was examined. 2. RS-(+/-) 2-(4-chlorophenoxy)propionic acid (RS-(+/-) CPP) and its S-(-) enantiomer produced pronounced effects on CIC-1 gating. Both compounds caused the channels to deactivate more rapidly at hyperpolarizing potentials, which showed as a decrease in the time constants of both the fast and slow deactivating components of the whole cell currents. Both compounds also produced a concentration-dependent shift in the voltage dependence of channel apparent open probability to more depolarizing potentials, with an EC50 of 0.79 and 0.21 mM for the racemate and S-(-) enantiomer respectively. R-(+) CPP at similar concentrations had no effect on gating. RS-(+/-) CPP did not block the passage of Cl- through the pore of rCIC-1. 3. CIC-1 is gated by Cl- binding to a site within an access channel and S-(-) CPP alters gating of the channel by decreasing the affinity of this binding site for Cl-. Comparison of the EC50 for RS-(+/-) CPP and S-(-) CPP indicates that R-(+) CPP can compete with the S-(-) enantiomer for the site but that it is without biological activity. 4. RS-(+/-) CPP produced the same effect on rCIC-1 gating when added to the interior of the cell and in the extracellular solution. 5. S-(-) CPP modulates the gating of CIC-1 to decrease the membrane Cl- conductance (GCl), which would account for the myotonic side effects of clofibrate and its derivatives.

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Using a large database of protein structure-structure alignments, we test a new method for distinguishing homologous and "analogous" structural neighbors. The homologous neighbors included in the test set show no detectable sequence similarity, but they may be well superimposed and show functional similarity or other evidence of evolutionary relationship. Analogous neighbors also show no sequence similarity and may be well superimposed, but they have different functions and their structural similarity may be the result of convergent evolution. Confirming results of other analyses, we find that remote homologs and analogs are not well distinguished by measures of pairwise structural similarity, including the percentage of identical residues and root-mean-square (RMS) superposition residual. We show, however, that with structure-structure alignments of analogous neighbors rarely superimpose the particular substructure that is shared among homologous neighbors. We call this characteristic substructure the homologous core structure (HCS), and we show that a cross-validated test for presence of the HCS correctly identifies 75% of remote homologs with a false-positive rate of 16% analogs, significantly better than discrimination by RMS or other measures of pairwise similarity. The HCS describes conservation of spatial structure within a protein family in much the way that a sequence motif describes sequence conservation. We suggest that it may be used in the same way, to identify homologous neighbors at greater evolutionary distance than is possible by pairwise comparison.

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The three dimensional structures for representatives of nearly half of all protein families are now available in public databases. Thus, no matter which protein one investigates, it is increasingly likely that the 3D structure of a homolog will be known and may reveal unsuspected structure-function relationships. The goal of Entrez's 3D-structure database is to make this information accessible and usable by molecular biologists (http://www.ncbi.nlm.nih.gov/Entrez). To this end Entrez provides two major analysis tools, a search engine based on sequence and structure 'neighboring' and an integrated visualization system for sequence and structure alignments. From a protein's sequence 'neighbors' one may rapidly identify other members of a protein family, including those where 3D structure is known. By comparing aligned sequences and/or structures in detail, using the visualization system, one may identify conserved features and perhaps infer functional properties. Here we describe how these analysis tools may be used to investigate the structure and function of newly discovered proteins, using the PTEN gene product as an example.

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A survey was compiled of several characteristics of the intersubunit contacts in 58 oligomeric proteins, and of the intermolecular contracts in the lattice for 223 protein crystal structures. The total number of atoms in contact and the secondary structure elements involved are similar in the two types of interfaces. Crystal contact patches are frequently smaller than patches involved in oligomer interfaces. Crystal contacts result from more numerous interactions by polar residues, compared with a tendency toward nonpolar amino acids at oligomer interfaces. Arginine is the only amino acid prominent in both types of interfaces. Potentials of mean force for residue-residue contacts at both crystal and oligomer interfaces were derived from comparison of the number of observed residue-residue interactions with the number expected by mass action. They show that hydrophobic interactions at oligomer interfaces favor aromatic amino acids and methionine over aliphatic amino acids; and that crystal contacts form in such a way as to avoid inclusion of hydrophobic interactions. They also suggest that complex salt bridges with certain amino acid compositions might be important in oligomer formation. For a protein that is recalcitrant to crystallization, substitution of lysine residues with arginine or glutamine is a recommended strategy.

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Prediction of protein structure by fold recognition, or threading, was recently put to the test in a 'blind' structure prediction experiment, CASP2. Thirty-two teams from around the world participated, preparing predictions for 22 different 'target' proteins whose structures were soon to be determined. As experimental structures became available, we, as organizers of the threading competition, computed objective measures of fold-recognition specificity and model accuracy, to identify and characterize successful predictions. Here, we present a brief summary of these prediction evaluations, a tally of 'correct' predictions and a discussion of factors associated with correct predictions. We find that threading produced specific recognition and accurate models whenever the structural database contained a template spanning a large fraction of target sequence. Presence of conserved sequence motifs was helpful, but not required, and it would appear that threading can succeed whenever similarity to a known structure is sufficiently extensive.

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Analysis of CASP2 protein threading results shows that the success rate of structure predictions varies widely among prediction targets. We set "critical" thresholds in fold recognition specificity and threading model accuracy at the points where "incorrect" CASP2 predictions just outnumber "correct" predictions. Using these thresholds we find that correct predictions were made for all of those targets and for only those targets where more than 50% of target residues may be superimposed on previously known structures. Three-fourths of these correct predictions were furthermore made for targets with greater than 12% residue identity in structural alignment, where characteristic sequence motifs are also present. Based on these observations we suggest that the sustained performance of threading methods is best characterized by counting the numbers of correct predictions for targets of increasing "difficulty." We suggest that target difficulty may be assigned, once the true structure of the target is known, according to the fraction of residues superimposable onto previously known structures and the fraction of identical residues in those structural alignments.

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Threading predictions for CASP2 target proteins were compared to their true structures using a series of precisely defined measures of agreement, calculated in a fully automatic way. Fold recognition specificity was calculated as the proportion of a predictor's "bet" that was placed on previously-known structures similar to the prediction target, as identified by a "jury" of well-tested structure-structure comparison methods. Values approaching 100% indicate that a prediction correctly identified the structural and/or evolutionary family to which a target belongs. Alignment specificity was calculated as the proportion of aligned residue paris in the predicted target-to-known-structure alignment that also occur in the structure-structure alignments produced by the "jury" methods. Contact specificity was calculated as the proportion of nonlocal residue contacts in the molecular model implied by threading alignment, that also occur in the experimental structure of the target. Alignment specificity and contact specificity measure the accuracy of a predicted 3-dimensional model. Values approaching 100% indicate that target residues have been assigned to the correct spatial locations and that the model is as accurate as possible for a threading prediction.

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Threading experiments with proteins from the globin family provide an indication of the nature of the structural similarity required for successful fold recognition and accurate sequence-structure alignment. Threading scores are found to rise above the noise of false positives whenever roughly 60% of residues from a sequence can be aligned with analogous sites in the structure of a remote homolog. Fold recognition specificity thus appears to be limited by the extent of structural similarity, regardless of the degree of sequence similarity. Threading alignment accuracy is found to depend more critically on the degree of structural similarity. Alignments are accurate, placing the majority of residues exactly as in structural alignment, only when superposition residuals are less than 2.5 A. These criteria for successful recognition and sequence-structure alignment appear to be consistent with the successes and failures of threading methods in blind structure prediction. They also suggest a direct assay for improved threading methods: Potentials and alignment models should be tested for their ability to detect less extensive structural similarities, and to produce accurate alignments when superposition residuals for this conserved "core" fall in the range characteristic of remote homologs.

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Examination of a protein's structural 'neighbors' can reveal distant evolutionary relationships that are otherwise undetectable, and perhaps suggest unsuspected functional properties. In the past, such analyses have often required specialized software and computer skills, but new structural comparison methods, developed in the past two years, increasingly offer this opportunity to structural and molecular biologists in general. These methods are based on similarity-search algorithms that are fast enough to have effectively removed the computer-time limitation for structure-structure search and alignment, and have made it possible for several groups to conduct systematic comparisons of all publicly available structures, and offer this information via the World Wide Web. Furthermore, and perhaps surprisingly given the difficulty of the structure-comparison problem, these groups seem to have converged on quite similar approaches with respect to both fast search algorithms and the identification of statistically significant similarities.

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We present an analysis of 10 blind predictions prepared for a recent conference, "Critical Assessment of Techniques for Protein Structure Prediction." The sequences of these proteins are not detectably similar to those of any protein in the structure database then available, but we attempted, by a threading method, to recognize similarity to known domain folds. Four of the 10 proteins, as we subsequently learned, do indeed show significant similarity to then-known structures. For 2 of these proteins the predictions were accurate, in the sense that a similar structure was at or near the top of the list of threading scores, and the threading alignment agreed well with the corresponding structural alignment. For the best predicted model mean alignment error relative to the optimal structural alignment was 2.7 residues, arising entirely from small "register shifts" of strands or helices. In the analysis we attempt to identify factors responsible for these successes and failures. Since our threading method does not use gap penalties, we may readily distinguish between errors arising from our prior definition of the "cores" of known structures and errors arising from inherent limitations in the threading potential. It would appear from the results that successful substructure recognition depends most critically on accurate definition of the "fold" of a database protein. This definition must correctly delineate substructures that are, and are not, likely to be conserved during protein evolution.

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