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S H Bryant

Publications and source records attributed to S H Bryant.

At least 19 recordsLinked to original sources
PubMed1995

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.

Amino Acid Sequence↗
PubMed1995

Homology model building of the HMG-1 box structural domain.

Nucleoproteins belonging to the HMG-1/2 family possess homologous domains approximately 75 amino acids in length. These domains, termed HMG-1 boxes, are highly structured, compact, and mediate the interaction between HMG-1 box-containing proteins and DNA in a variety of biological contexts. Homology model building experiments on HMG-1 box sequences 'threaded' through the 1H-NMR structure of an HMG-1 box from rat indicate that the domain does not have rigid sequence requirements for its formation. Energy calculations indicate that the structure of all HMG-1 box domains is stabilized primarily through hydrophobic interactions. We have found structural relationships in the absence of statistically significant sequence similarity, identifying several candidate proteins which could possibly assume the same three-dimensional conformation as the rat HMG-1 box motif. The threading technique provides a method by which significant structural similarities in a diverse protein family can be efficiently detected, and the 'structural alignment' derived by this method provides a rational basis through which phylogenetic relationships and the precise sites of interaction between HMG-1 box proteins and DNA can be deduced.

Amino Acid Sequence↗
PubMed1995

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.

Algorithms↗
PubMed1995

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.

Algorithms↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

Effects of veratridine on Na and Ca currents in frog skeletal muscle.

1. Voltage-clamp experiments were performed to determine the effects of veratridine on Na and Ca currents in frog skeletal muscle fibres. 2. Veratridine (1 microM) did not affect the kinetics of the fast Na current but it did induce a slowly inactivating tetrodotoxin-sensitive inward current that was apparent after Na current inactivation. This slow current had a peak amplitude of 6.7 +/- 0.7 microA/cm2 at -20 mV and decayed monoexponentially with a time constant of 606 +/- 77 ms. 3. The slow current had a voltage-dependence for activation that was similar to that of the fast Na current. Single depolarizing prepulses that induced complete inactivation of the fast Na channels, prevented development of the slow current. Trains of brief depolarizations at increasing frequencies increased the amplitude of the slow current. These results suggest that the slow current may be mediated by veratridine modified Na channels that must be in the open position. 4. The low concentration of veratridine (1 microM) did not affect the Ca current, while 100 microM veratridine reversibly suppressed the Ca current and shifted its peak current-voltage relation towards more negative potentials. Thus, veratridine appears not to be a selective fast Na channel modifier as it may also alter Ca channel gating properties in skeletal muscle fibres.

Animals↗
PubMed1994

Effects of veratrine on ion currents in single rabbit cardiomyocytes.

1. Voltage-clamp experiments were performed to determine the effects of veratrine (1 microgram/ml) on Na and K currents in isolated rabbit ventricular cardiomyocytes. 2. Veratrine did not affect the inward rectifier K current, increased the inactivation time constant of the transient outward current (I(to)) and induced a slowly decaying inward current component (Iv), which was sensitive to tetrodotoxin. 3. Inactivation of fast Na channels by application of short depolarizing prepulses to potentials between -90 and -50 mV prevented the development of Iv.Iv decayed biexponentially with time constants equal to 139 +/- 9.0 ms and 776 +/- 47 ms. The net amplitude of Iv and the time constants for its rapidly and slowly inactivating components were little affected by trains of conditioning prepulses to 0 mV. The contributions, however, of the fast and slow components to the net current were significantly altered by repetitive depolarizations. 4. These components of Iv are likely due to modification of open cardiac Na channels by veratrum alkaloids.

Animals↗
PubMed1994

An electrophysiological survey of frog olfactory cilia.

Individual olfactory receptor neurons vary widely in their responses to odorants. Olfactory stimulus reception occurs in the cilia of the receptor neurons. Thus, the variability among individual neurons could in part be due to differences among the olfactory cilia. We have quantified the known conductance properties of each of 117 frog olfactory cilia. From a strictly qualitative viewpoint, the cilia were very homogeneous. All but a few of them had a basal conductance in the absence of odorants and second messengers, conductances stimulated by cytoplasmic cyclic AMP and by Ca2+ and a conductance measured in the presence of ATP and stimulated by GTP gamma S. However, the magnitudes of the conductances varied widely among the cilia. Amplitudes of the cyclic-AMP- and Ca(2+)-activated ciliary currents correlated strongly with one another across the 117 cilia and 24 frogs studied, suggesting that expression of the underlying channels may be co-regulated. None of the conductance properties correlated strongly with ciliary length, a marker of cell maturity. Given cytoplasmic MgATP as substrate, ciliary adenylate cyclase apparently produced cyclic AMP, which in turn gated membrane channels and increased the ciliary conductance. In some cilia, MgATP alone caused a very large increase in conductance. In others, there was little effect unless GTP gamma S, which increases cyclase activity, was also added. No effect of cytoplasmic inositol trisphosphate on ciliary conductance was detectable.

Adenosine Triphosphate↗
PubMed1993

An empirical energy function for threading protein sequence through the folding motif.

In this paper we present a new residue contact potential derived by statistical analysis of protein crystal structures. This gives mean hydrophobic and pairwise contact energies as a function of residue type and distance interval. To test the accuracy of this potential we generate model structures by "threading" different sequences through backbone folding motifs found in the structural data base. We find that conformational energies calculated by summing contact potentials show perfect specificity in matching the correct sequences with each globular folding motif in a 161-protein data set. They also identify correct models with the core folding motifs of hemerythrin and immunoglobulin McPC603 V1-domain, among millions of alternatives possible when we align subsequences with alpha-helices and beta-strands, and allow for variation in the lengths of intervening loops. We suggest that contact potentials reflect important constraints on nonbonded interaction in native proteins, and that "threading" may be useful for structure prediction by recognition of folding motif.

Amino Acid Sequence↗
PubMed1993

Regulation of resting ionic conductances in frog skeletal muscle.

The membrane electrical properties and resting ionic conductances of frog semitendinosus muscle fibres were studied in vitro at 25 degrees C with the two-micro-electrode cable technique, in the presence of an activator or inhibitor of protein kinase C (PKC) or in the presence of an activator of adenylate cyclase. The PKC activator, 4 beta-phorbol 12,13-dibutyrate (4 beta-PDB), reduced chloride conductance (GCl) at concentrations greater than 1 microM and did not affect potassium conductance (GK). At 150 microM, the maximum concentration of 4 beta-PDB tested, GCl was reduced by 42%. The "inactive" phorbol ester 4 alpha-phorbol 12,13-dibutyrate did not affect GCl or GK. The inhibitory effect of 4 beta-PDB on GCl was prevented by pretreatment of the muscle preparation with the PKC inhibitor staurosporine. The adenylate cyclase activator forskolin (1.5-8 microM) significantly increased the GK of the fibres, without affecting GCl. Thus, we conclude that frog skeletal muscle GCl, unlike rat muscle GCl, is relatively insensitive to activators of PKC. Moreover, in frog muscle, protein kinase A is a likely modulator of GK, but not GCl.

Adenylyl Cyclases↗
PubMed1993

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.

Animals↗
PubMed1992

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.

Animals↗
PubMed1992

Opposite effects of enantiomers of clofibric acid derivative on rat skeletal muscle chloride conductance: antagonism studies and theoretical modeling of two different receptor site interactions.

The R-(+) enantiomer of 2-(p-chlorophenoxy) propionic acid (CPP) produces a biphasic effect on chloride channel conductance (GCl) of rat skeletal muscle, increasing GCl at low concentrations and decreasing it at concentrations greater than 10 microM; on the other hand, the S-(-) isomer mostly blocks GCl in a concentration-dependent manner. To explain the peculiar behavior of these compounds, a theoretical model based on the presence of two opposing receptor populations controlling chloride channel conductance has been used to fit the experimental data of the concentration-response curves of both S-(-) and R-(+) CPP. An analysis performed by means of the algebraic summation of two logistic terms suggests a reasonable merit of the proposed model and explains the resultant effect of each optical form as follows: S-(-) acts as a full agonist on an inhibitory sites, whereas R-(+) acts as a full agonist at both the inhibitory and excitatory sites. Antagonism studies appear to be consistent with the proposed model. Dose-response curves in which the block of GCl by the S-(-) isomer was evaluated in the presence of the R-(+) isomer (3-10 microM) clearly showed an antagonistic interaction between the two enantiomers, with an increase in the S-(-) concentration for half-maximal block. The antagonism was overcome by high concentrations of S-(-), and this might be consistent with the hypothesis that the block of GCl is modulated by an inhibitory site at which the two enantiomers compete.

2-Methyl-4-chlorophenoxyacetic Acid↗
PubMed1991

Four decades of inversion polymorphism in Drosophila pseudoobscura.

We report data that continue the studies of Dobzhansky and others on the frequencies of third-chromosome inversions in natural populations of Drosophila pseudoobscura in North America. The common gene arrangements continue to be present in frequencies similar to those described four decades ago, and the broad geographic patterns also remain unchanged. There is only one pronounced trend over time: the increase in frequency of the Tree Line inversion in Pacific coast populations.

Animals↗
PubMed1991

The frequency of ion-pair substructures in proteins is quantitatively related to electrostatic potential: a statistical model for nonbonded interactions.

A statistical analysis of ion pairs in protein crystal structures shows that their abundance with respect to uncharged controls is accurately predicted by a Boltzmann-like function of electrostatic potential. It appears that the mechanisms of protein folding and/or evolution combine to produce a "thermal" distribution of local nonbonded interactions, as has been suggested by statistical-mechanical theories. Using this relationship, we develop a maximum likelihood methodology for estimation of apparent energetic parameters from the data base of known structures, and we derive electrostatic potential functions that lead to optimal agreement of observed and predicted ion-pair frequencies. These are similar to potentials of mean force derived from electrostatic theory, but departure from Coulombic behavior is less than has been suggested.

Biological Evolution↗
PubMed1991

Chloride channel regulation in the skeletal muscle of normal and myotonic goats.

External intercostal muscle biopsies from normal and congenitally myotonic goats were studied in vitro at 30 degrees C using a two-microelectrode square-pulse cable analysis assisted by computer. The resting chloride conductance (Gcl) was estimated from the difference between the mean membrane conductance in chloride-containing and chloride-free bathing media. The protein kinase C (PKC) activator, 4-beta-phorbol-12,13-dibutyrate. (0.1-2.0 microM) blocks a maximum of 76% of Gcl in normal goat fibers and induces myotonic hyperexcitability similar to that of congenitally myotonic goat fibers. The Gcl block was partially antagonized by pretreatment with the PKC inhibitor, staurosporine (10 microM). The "inactive" 4-alpha-phorbol-12,13-didecanoate had no effect at 50 microM, whereas the "active" 4-beta isomer blocked 41% Gcl at 1 microM. The nearly absent Gcl of congenitally myotonic goat fibers was not restored by treatment with high concentrations of the PKC inhibitors staurosporine, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), or tetrahydropapaveralone (THP). Also, forskolin and cholera toxin, which may increase cyclic adenosine monophosphate (cAMP) levels, or the R(+) clofibric acid enantiomers and taurine, which increase Gcl in normal fibers, were also unable to restore Gcl in myotonic goat fibers. The data suggest that PKC may be a chloride channel regulator in normal goat skeletal muscle fibers, however the molecular defect of congenitally myotonic fiber does not appear to be due to excessive activity of PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Modulation of rat skeletal muscle chloride channels by activators and inhibitors of protein kinase C.

The membrane electrical parameters and component conductances of rat extensor digitorum longus muscle fibres were studied in vitro at 30 degrees C with standard two microelectrode square pulse cable analysis in the presence of protein kinase C (PKC) activators and inhibitors. The PKC activator, 4-beta-phorbol-12,13 dibutyrate (4-beta-PDB), (2-90 nM) blocked up to 67% chloride conductance (GCl) in rat skeletal muscle fibres and induced myotonic hyperexcitability. The concentration necessary to produce a 50% block of the membrane GCl was 23 nM. The "inactive" 4-alpha-phorbol-12,13 dibutyrate had no effect at 2 microM. The blocking effect of 4-beta-PDB on GCl was prevented by preincubation of the preparations with the PKC inhibitors, staurosporine (1-5 microM) and tetrahydropapaverolone (50-100 microM). The blocking effects on membrane GCl of 4-beta-PDB and its antagonism by the inhibitors used support the concept of the involvement of PKC in regulating Cl channels of mammalian skeletal muscle fibres.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗