A measure of success in fold recognition.

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.

Cable parameters, sodium, potassium, chloride, and water content, and potassium efflux in isolated external intercostal muscle of normal volunteers and patients with myotonia congenita.

In isolated fiber bundles of external intercostal muscle from each of 13 normal volunteers and each of 6 patients with myotonia congenita, some or all of the following were measured: concentrations of Na(+), K(+), and Cl(-), extracellular volume, water content, K(+) efflux, fiber size, fiber cable parameters, and fiber resting potentials. Muscle from patients with myotonia congenita differed significantly (0.001

Statistics of sequence-structure threading.

The past two years have seen the rapid development of new recognition methods for protein structure prediction. These algorithms 'thread' the sequence of one protein through the known structure of another, looking for an alignment that corresponds to an energetically favorable model structure. Because they are based on energy calculation, rather than evolutionary distance, these methods extend the possibility of structure prediction by comparative modeling to a larger class of new sequences, where similarity to known structures is recognizable by no other means. The strength of the evidence they offer should be judged by objective statistical tests, however, so as to rule out the possibility that favorable scores arise from chance factors such as similarity of length, composition, or the consideration of a large number of alternative alignments. Calculation of objective p-values by analytical means is not yet possible, but it would appear that approximate values may be obtained by simulation, as they are in gapped, global sequence alignment. We propose that the results of threading experiments should include Z-scores relative to the composition-corrected score distribution obtained for shuffled and optimally aligned sequences.

Surprising similarities in structure comparison.

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.

Sodium, potassium, and chloride fluxes in intercostal muscle from normal goats and goats with hereditary myotonia.

IN ISOLATED BUNDLES OF EXTERNAL INTERCOSTAL MUSCLE FROM NORMAL GOATS AND GOATS WITH HEREDITARY MYOTONIA THE FOLLOWING WERE DETERMINED: concentrations and unidirectional fluxes of Na(+), K(+), and Cl(-), extracellular volume, water content, fiber geometry, and core-conductor constants. No significant difference between the two groups of preparations was found with respect to distribution of fiber size, intracellular concentrations of Na(+) or Cl(-), fiber water, resting membrane potential, or overshoot of action potential. The intracellular Cl(-) concentration in both groups of preparations was 4 to 7 times that expected if Cl(-) were distributed passively between intracellular and extracellular water. The membrane permeability to K (P(K)) calculated from efflux data was (a) at 38 degrees C, 0.365 x 10(-6) cm sec(-1) for normal and 0.492 x 10(-6) for myotonic muscle, and (b) at 25 degrees C, 0.219 x 10(-6) for normal and 0.199 x 10(-6) for myotonic muscle. From Cl(-) washout curves of normal muscle usually only three exponential functions could be extracted, but in every experiment with myotonic muscle there was an additional, intermediate component. From these data PP(cl) could be calculated; it was 0.413 x 10(-6) cm sec(-1) for myotonic fibers and was 0.815 x 10(-6) cm sec(-1) for normal fibers. The resting membrane resistance of myotonic fibers was 4 to 6 times greater than that of normal fibers.

Combination of threading potentials and sequence profiles improves fold recognition.

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.

Clan lethals and inter-year allelism in Death Valley Drosophila pseudoobscura.

A chromosome 2 lethal allelism rate of about 3% found in the 1974 population of D. pseudoobscura in Death Valley, California. This rate was significantly higher than allelism rates in other Southern California populations. The Death Valley population was sampled again in 1975 and 1977, with allelism rates of 1% and 0.5%, respectively. In 1974, several lethals were in high frequencies (about 1%), a pattern that reappeared in 1975 and 1977. However, none of the lethals in high frequency one year was in high frequency another year; the particular lethal alleles present in this ephemeral population appear to be due to their random presence in the flies which refound the population every winter. The results for the Death Valley population are compared with a Japanese population of D. melanogaster in which lethals in high frequency one year also in high frequency in succeeding years and with earlier work on chromosome 3 of D. pseudoobscura, which showed a lower lethal frequency and higher allelism rate.

The frequency and allelism of lethal chromosomes in isolated desert populations of Drosophila pseudoobscura.

Second-chromosome lethals were extracted from four populations of Drosophila pseudoobscura in Southern California. Two of the populations were from desert oases and two from the classic habitat on Mt. San Jacinto, previously studied by Dobzhansky. Allelism tests were made on the lethals within and between all locations. The frequency of lethal second-chromosomes in each location was 0.18, and this was not different from the results of other workers for samples throughout the species range. Interpopulational allelism rates were about 0.005, and not different from earlier results of Dobzhansky. Intrapopulational rates in this study were, with one exception, the same as the interpopulational rates, and significantly lower than Dobzhansky found using the third chromosome. This may be due to lethals being linked with heterotic third-chromosome inversions. The allelism rate of the exceptional population (about 0.03 and equal to Dobzhansky's intrapopulational results) may be due to heterotic lethals, or a founder effect. Two lethals were found in three populations each, possibly due to migration among these populations, which are up to 334 km apart.

Gene flow and the geographical distribution of a molecular polymorphism in Drosophila pseudoobscura.

This paper discusses the relation between the geographical distribution of an enzyme polymorphism and population structure in Drosophila pseudoobscura. California populations of this species living in very different montane and lowland habitats separated by several kilometers are similar to each other in the frequency of an esterase allele. Previous estimates suggest that gene flow is too limited to account for this homogeneity of genetic structure, so that it must reflect some balancing force of natural selection. We slow, however, that dispersal over unfavorable habitats is much greater than earlier supposed. Isolated populations of D. pseudoobscura separated by 15 km from other populations are subject to large amounts of immigration. This is shown by changes in the seasonal abundance of this species and in the annual pattern of lethal alleles in such populations. The genetic structure of an experimentally perturbed isolated population in an oasis returned to normal within a single year, suggesting that such populations are ephemeral and that the oasis is subject to annual recolonization by distant migrants. Direct assessment of marked flies shows that they can move at least 10 10 km in 24 hours over a desert. Such extensive gene flow may help explain the distribution of the esterase allele, and is relevant to the high level of molecular polymorphism and its general lack of geographic differentiation throughout the range of D. pseudoobscura.

A proposed structural model of domain 1 of fasciclin III neural cell adhesion protein based on an inverse folding algorithm.

Fasciclin III is an integral membrane protein expressed on a subset of axons in the developing Drosophila nervous system. It consists of an intracellular domain, a transmembrane region, and an extracellular region composed of three domains, each predicted to form an immunoglobulin-like fold. The most N-terminal of these domains is expected to be important in mediating cell-cell recognition events during nervous system development. To learn more about the structure/function relationships in this cellular recognition molecule, a model structure of this domain was built. A sequence-to-structure alignment algorithm was used to align the protein sequence of the fasciclin III first domain to the immunoglobulin McPC603 structure. Based on this alignment, a model of the domain was built using standard homology modeling techniques. Side-chain conformations were automatically modeled using a rotamer search algorithm and the model was minimized to relax atomic overlaps. The resulting model is compact and has chemical characteristics consistent with related globular protein structures. This model is a de novo test of the sequence-to-structure alignment algorithm and is currently being used as the basis for mutagenesis experiments to discern the parts of the fasciclin III protein that are necessary for homophilic molecular recognition in the developing Drosophila nervous system.

Eukaryotic translation elongation factor 1 gamma contains a glutathione transferase domain--study of a diverse, ancient protein superfamily using motif search and structural modeling.

Using computer methods for multiple alignment, sequence motif search, and tertiary structure modeling, we show that eukaryotic translation elongation factor 1 gamma (EF1 gamma) contains an N-terminal domain related to class theta glutathione S-transferases (GST). GST-like proteins related to class theta comprise a large group including, in addition to typical GSTs and EF1 gamma, stress-induced proteins from bacteria and plants, bacterial reductive dehalogenases and beta-etherases, and several uncharacterized proteins. These proteins share 2 conserved sequence motifs with GSTs of other classes (alpha, mu, and pi). Tertiary structure modeling showed that in spite of the relatively low sequence similarity, the GST-related domain of EF1 gamma is likely to form a fold very similar to that in the known structures of class alpha, mu, and pi GSTs. One of the conserved motifs is implicated in glutathione binding, whereas the other motif probably is involved in maintaining the proper conformation of the GST domain. We predict that the GST-like domain in EF1 gamma is enzymatically active and that to exhibit GST activity, EF1 gamma has to form homodimers. The GST activity may be involved in the regulation of the assembly of multisubunit complexes containing EF1 and aminoacyl-tRNA synthetases by shifting the balance between glutathione, disulfide glutathione, thiol groups of cysteines, and protein disulfide bonds. The GST domain is a widespread, conserved enzymatic module that may be covalently or noncovalently complexed with other proteins. Regulation of protein assembly and folding may be 1 of the functions of GST.

Threading analysis suggests that the obese gene product may be a helical cytokine.

The ob gene encodes a protein that, in mutant form, is associated with obesity and type II diabetes in mice. Sequence analysis has revealed no similarities to other proteins, however, and no clues as to possible functions. The possibility nonetheless remains that ob is functionally or ancestrally related to other proteins, whose sequences are divergent to the point that only a comparison of three-dimensional structures might detect relationship. To explore this possibility, we conduct a 'threading' search of a 3-dimensional structure database, to determine whether the ob protein might adopt a fold similar to any known structure. This search reveals that the ob sequence is compatible, at a significance level of P < 0.05, with structures from the family of helical cytokines that includes interleukin-2 and growth hormone. A structural model of ob based upon these results is physically and biologically plausible and leads to testable predictions, including the prediction that ob may activate the JAK-STAT pathway, via binding to a receptor resembling those of the cytokine family.

Antimyotonic effects of tocainide enantiomers on skeletal muscle fibers of congenitally myotonic goats.

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.

Stereospecificity of the chloride ion channel: the action of chiral clofibric acid analogues.

2-(p-Chlorophenoxy)isobutyric acid (clofibric acid (1) or CPIB) is a drug known to block chloride membrane conductance (GCl) in rat striated muscle. In the present study chiral analogues of CPIB (2-(p-chlorophenoxy)propionic acid (2) and 2-(p-chlorophenoxy)butyric acid (3)) have been tested to evaluate the influence of chirality on Cl ion flux in the channel. The results showed that the chloride channel conductance strongly depends on the absolute configuration: in fact, the S-(-) isomers of the tested compounds strongly decreased the GCl of skeletal muscle membrane, whereas the R-(+) isomers were virtually ineffective. These data allow the hypothesis that, like other ion channels present in various biological systems, the chloride channel of skeletal muscle membrane could also have a stereospecific binding site (or receptor) regulating chloride ion flux.

Dissociation of hypolipidemic and antiplatelet actions from adverse myotonic effects of clofibric acid related enantiomers.

Enantiostructure-activity studies of chlorophenoxybutyric and propionic acids have provided evidence for the dissociation of serum cholesterol lowering and platelet antiaggregatory activities from the adverse chloride ion channel mediated myotonic effects of these compounds. R-(+) propionic and butyric acid enantiomers, unlike achiral clofibric acid and the S-(-) isomers, did not inhibit chloride conductance in rat extensor digitorum longus muscle fibers in vitro but, like clofibric acid and the S-(-) isomers, retained the serum cholesterol lowering activity in a cholesterol-fed rat model. Additionally, a stereoselective and greater inhibition was observed for the R-(+) isomers against adenosine diphosphate and arachidonic acid induced human platelet aggregation.

Modulation of the gating of CIC-1 by S-(-) 2-(4-chlorophenoxy) propionic acid.

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.

Topographic analysis of human follicle-stimulating hormone-beta using anti-peptide antisera.

The purpose of this study was to identify peptide sequences of human follicle-stimulating hormone-beta (hFSH beta) which are accessible subsequent to association with hFSH alpha in heterodimeric hFSH. Antisera were raised against synthetic peptides (Abpep) corresponding to hFSH beta sequences 1-20, 16-36, 33-53, 49-67, 66-85, 81-100 and 98-111. The topography of hFSH beta was studied by testing the binding of these antisera to hFSH beta and hFSH captured by monoclonal antibodies (MAb) in an enzyme-linked immunosorbent assay (ELISA). When hFSH and hFSH beta were captured by the same MAb, binding of Ab16-36, Ab33-53, Ab81-100 and Ab98-111 to hFSH was significantly lower compared to hFSH beta. However, compared to other Abpep, binding of Ab35-53 to hFSH was strong. Similar results were obtained when hFSH was captured by an alpha-specific MAb (10.3A6). Using 10.3A6, it was also possible to demonstrate significant binding of Ab49-67 to hFSH. The data suggests that residues in regions 33-53 and 49-67 of hFSH beta appear to be accessible in the heterodimeric hFSH in addition to the glycosylated region of 1-15. Regions 16-36, 33-53, 81-100 and 98-111 of hFSH beta appear to contain subunit contact-associated sequences which are either masked or structurally altered subsequent to association with hFSH alpha in the heterodimeric hFSH.

New programs for protein tertiary structure prediction.

Prediction of protein tertiary structure remains an unsolved problem in molecular biology, but a solution to this problem is extremely important for protein engineering and rational drug design. Recent developments in motif recognition and side chain modeling present the prospect of nearly automatic model building for a large fraction of newly determined protein sequences. We review some of these new algorithms and present preliminary results of their application to the prediction of a structure for fasciclin III, a neural adhesion molecule from Drosophila.