Search

Intrinsically disordered tails are common in DNA-binding proteins and can affect their search efficiency on nonspecific DNA by promoting the brachiation dynamics of intersegment transfer. During brachiation, the protein jumps between distant DNA regions via an intermediate state in which the tail and globular moieties are bound to different DNA segments. While the disordered tail must be long and positively charged to facilitate DNA search, the effect of its residue sequence on brachiation is unknown. We explored this issue using the NK-2 and Antp homeodomain transcription factors. We designed 566 NK-2 tail-variants and 55 Antp tail-variants having different net charges and positive charge distributions and studied their dynamics and DNA search efficiencies using coarse-grained molecular dynamics simulations. More intersegment transfers occur when the rail is moderately positively charged and the positive charges are clustered together in the middle of the tail or towards its N terminus. The presence of a negatively charged residue does not significantly affect protein brachiation, although it is likely that the presence of many negatively charged residues will complicate the DNA search mechanism. A bioinformatic analysis of 1,384 wild-type homeodomains illustrates that the charge composition and distribution in their N-tail sequenceS are consistent with an optimal charge pattern to promote intersegment transfer. Our study thus indicates that the residue sequence of the disordered tails of DNA-binding proteins has unique characteristics that were evolutionarily selected to achieve optimized function and suggests that the sequence-structure-function paradigm known for structured proteins is valid for intrinsically disordered proteins as well. intrinsically disordered proteins | protein-DNA interaction | sliding | intersegment transfer | Monkey-bar mechanism doi/ 10.1073/pnas.1011775107

For many aspects of DNA-protein interaction, it is vital to know how DNA bending rigidity (or persistence length, a) depends on its sequence. We addressed this problem using the method based on cyclization of short DNA fragments, which allows very accurate determination of a. Our approach was based on assigning specific values of a to each of 10 distinct dinucleotide steps. We prepared DNA fragments, each about 200 bp in length, with various quasi-periodic sequences, measured their cyclization efficiencies (j factors), and fitted the data by the theoretical equation to obtain the values of a for each fragment. From these data, we obtained a set of a for the dinucleotide steps. To test this set, we used it to design DNA sequences that should correspond to very low and very high values of a, prepared the corresponding fragments, and determined their values of a experimentally. The measured and calculated values of a were very close to one another, confirming that we have found the correct solution to this long-standing problem. The same experimental data also allowed us to determine the sequence dependence of DNA helical repeat. DNA elasticity | DNA persistence length doi/ 10.1073/pnas.1004809107

Sequencing of the genome of Clostridium botulinum strain Hall A revealed a gene (CBO0515), whose putative amino acid sequence was suggestive of the rare enzyme [N.sup.5]-(1-carboxyethyl) ornithine synthase. To test this hypothesis, CBO0515 has been cloned, and the encoded polypeptide was purified and characterized. This unusual gene appears to be confined to proteolytic strains assigned to group 1 of C. botulinum. doi:10.1128/JB.01044-09

It is demonstrated that, properly represented, the amino acid composition of protein sequences contains the information necessary to delineate the global properties of protein structure space. A numerical representation of amino acid sequence in terms of a set of property factors is used, and the values of those property factors are averaged over individual sequences and then over sets of sequences belonging to structurally defined groups. These sequence sets then can be viewed as points in a 10-dimensional space, and the organization of that space, determined only by sequence properties, is similar at both local and global scales to that of the space of protein structures determined previously. proteomics | sequence analysis | sequence-structure relationship

Ricinus communis (also know as the castor bean piano whose forbears escaped from suburban gardens or commercial cultivation grow wild in many countries. In temperate and tropical climates seeds will develop to maturity, and plants may be perennial. In Australia these plants have become widespread and are regarded as noxious weeds in many localities. The seeds of R. communis contain ricin, a protein toxin which can easily be extracted into an aqueous solution. Ricin is toxic by ingestion, inhalation, and injection. The history of terrorist and anarchist interest in the use of seeds from R. communis has driven the development of strategies for determination of cultivar and geographic location of the source of an extract of wild-grown castor bean seed. This forensic information is of considerable interest to law enforcement and intelligence organizations. During forensic studies of both the metabolome and proteome of extracts from eight specimens of six different cultivars of R. communis ('zanzibariensis' collected from Kenya and Tanzania, 'gibsonii', 'impala', 'dehradun', 'carmencita', and 'sanguineus' collected from Spain and Tanzania), three peptide biomarkers (designated Ricinus communis biomarkers, or RCB) were identified in both the MALDI and electrospray LC-MS spectra. Two of these peptides (RCB-1 and RCB-2) were present in varying amounts in all cultivars, while RCB-3 was present only in the 'carmencita' cultivar. The amino acid sequences of RCB-1 to -3 were determined using LC-M[S.sup.n] fragmentation and de novo sequencing on both the intact and the carbamidomethyl modified peptides. The connectivity of the two disulfide bonds that were present in all three RCB were determined using a strategy of partial reduction and differential alkylation using tris(2-carboxyethyl)phosphine with N-ethylmaleimide to reduce and alkylate the most accessible disulfide bond, followed by reduction and alkylation of the remaining disulfide bond with dithiolthreitol and iodoacetamide. The possible functional role of RCB-1 to -3 in R. communis seeds is also discussed.

Comprehensive phosphorylation site mapping is the central goal of phosphoproteome studies, but complete protein sequence coverage is rarely obtained using one single protease. In this study, we have evaluated the use of elastase, in comparison to trypsin, to increase phosphorylation site coverage of mitotic spindle proteins enriched from cultured human ceils. We took advantage of the high mass accuracy of Orbitrap mass spectrometers and optimized the database search specificity by analyzing both elastase cleavage preferences and employing a dedicated two-step database search strategy. Through this approach, we have approximately doubled the number of detectable phosphorylation sites from elastase digested samples. Remarkably, phosphorylation sites detected by trypsin and elastase were highly complementary with an overlap of less than 10%. In total, we identified 1068 phosphorylation sites using trypsin and 467 phosphorylation sites using elastase. Approximately 30% of the phosphorylation sites were exclusively identified after digestion by elastase, demonstrating the value of this enzyme for phosphoproteome studies.

The structures of mitochondrial transporters and uncoupling proteins are 3-fold pseudosymmetrical, but their substrates and coupling ions are not. Thus, deviations from symmetry are to be expected in the substrate and ion-binding sites in the central aqueous cavity. By analyzing the 3-fold pseudosymmetrical repeats from which their sequences are made, conserved asymmetric residues were found to cluster in a region of the central cavity identified previously as the common substrate-binding site. Conserved symmetrical residues required for the transport mechanism were found at the water--membrane interfaces, and they include the three PX[DE]XX[RK] motifs, which form a salt bridge network on the matrix side of the cavity when the substrate-binding site is open to the mitochondrial intermembrane space. Symmetrical residues in three [FY][DE]XX[RK] motifs are on the cytoplasmic side of the cavity and could form a salt bridge network when the substrate-binding site is accessible from the mitochondrial matrix. It is proposed that the opening and closing of the carrier may be coupled to the disruption and formation of the 2 salt bridge networks via a 3-fold rotary twist induced by substrate binding. The interaction energies of the networks allow members of the transporter family to be classified as strict exchangers or uniporters. membrane proteins | substrate binding | amino acid sequence analysis I salt bridge networks | conformational changes

Inferring evolutionary relationships among highly divergent protein sequences is a daunting task. In particular, when pairwise sequence alignments between protein sequences fall ab initio | retroelements | reverse transcriptase | GDDA-BLAST

A de novo chromosome 7q inversion disrupting Autism susceptibility candidate 2 (AUTS2) and Contactin Associated Protein-Like 2 (CNTNAP2) in a child with congitive and social delay is identified. It is suggested that rare variants contribute to the pathophysiology of autism spectrum disorders (ASD).

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a food-borne pathogen that causes hemorrhagic colitis and the hemolytic uremic syndrome. Colonization of the human gut mucosa and production of potent Shiga toxins are critical virulence traits of EHEC. Although EHEC O157:H7 contains numerous putative pill operons, their role in the colonization of the natural bovine or accidental human hosts remains largely unknown. We have identified in EHEC an adherence factor, herein called E. coli common pilus (ECP), composed of a 21-kDa pilin subunit whose amino acid sequence corresponds to the product of the yagZ (renamed ecpA) gene present in all E. coli genomes sequenced to date. ECP production was demonstrated in 121 (71.6%) of a total of 169 [ecpA.sup.+] strains representing intestinal and extraintestinal pathogenic as well as normal flora E. coli. High-resolution ultrastructural and immunofluorescence studies demonstrated the presence of abundant peritrichous fibrillar structures emanating from the bacterial surface forming physical bridges between bacteria adhering to cultured epithelial cells. Isogenic ecpA mutants of EHEC O157:H7 or fecal commensal E. coli showed significant reduction in adherence to cultured epithelial cells. Our data suggest that ECP production is a common feature of E. coli colonizing the human gut or other host tissues. ECP is a pilus of EHEC O157:H7 with a potential role in host epithelial cell colonization and may represent a mechanism of adherence of both pathogenic and commensal E. coli. pill | enterohemorrhagic Escherichia coli | normal flora

Study is conducted to compare the inhibition kinetics of a hydroxamate based inhibitor of ADAMTS-4 using a small peptide substrate versus the native substrate, aggrecan. It also defines the mode of inhibition of the ADAMTS-4 inhibitor with both substrates.

A study presents the evidence for a regulatory subunit that preferentially interacts with protein phosphatase-1c[gamma]2 (PP1cgamma2) and has the potential to target the isoform to a function required for spermatogenesis. The isolation of another isoform specific regulatory subunit, Spzl (22), is identified in the same yeast two-hybrid screen as endophilin Blt.d

The studies show that both K600 in hs mt LeuRS and L570 in its bacterial homologue Escherichia coli through evolution is strategically positioned and carefully chosen at these corresponding locations for maximum overall efficiency and balance in both amino acid discrimination and tRNA binding. It is also found that E. coli glutaminyl-tRNA synthetase balances substrate specificity with catalytic efficiency.

A newly designed amino acid sequence containing one tyrosine residue is employed to study and describe the various proton-coupled electron-transfer reactions in a biomimetic peptide, which are often used as a model for the study of the enzyme regulatory mechanisms in proteins. The results of the analysis show that the thermodynamic coupling increases the yield of the tyrosyl radical at low pH by stabilizing it due to the interstrand [pi]-cation interaction.

A series of molecular and biochemical analyses were used to compare the nature of the structural elements of h- and mCG-[beta] subunits which is responsible for differences in conformational freedom between the respective CG-[beta]-subunits. The results show that the two specific amino acid residues identified in a four amino-acid subdomain regulate CG-[beta] conformation freedom.

Nearly 60 cell adhesive peptides derived from laminin-1 were screened for identification of additional amyloidogenic sequences. The results show that at least five laminin derived peptides can form amyloid-like fibrils, thus concluding that the laminin derived amyloidogenic peptides have the potential to form amyloid-like fibrils in vivo when laminin-1 is degraded.

In this article, we develop an evolutionary model for protein sequence evolution. Gene pleiotropy is characterized by K distinct but correlated components (molecular phenotypes) that affect the organismal fitness. These K molecular phenotypes are under stabilizing selection with microadaptation (SM) due to random optima shifts, the SM model. Random coding mutations generate a correlated distribution of K molecular phenotypes. Under this SM model, we further develop a statistical method to estimate the 'effective' number of molecular phenotypes ([K.sub.e]) of the gene. Therefore, for the first time we can empirically evaluate gene pleiotropy from the protein sequence analysis. Case studies of vertebrate proteins indicate that [K.sub.e] is typically ~6-9. We demonstrate that the newly developed SM model of protein evolution may provide a basis for exploring genomic evolution and correlations.

Two models of the origin of novel polypeptide domains are proposed, based on 'exonization' of intron sequences and insertion-based polymorphisms between orthologous genes. These processes are discussed, also analyzing how each might participate in the evolutionary emergence of novel polypeptide domains.

As an important model for transmembrane signaling, methylaccepting chemotaxis proteins (MCPs) have been extensively studied by using genetic, biochemical, and structural techniques. However, details of the molecular mechanism of signaling are still not well understood. The availability of genomic information for hundreds of species enables the identification of features in protein sequences that are conserved over long evolutionary distances and thus are critically important for function. We carried out a large-scale comparative genomic analysis of the MCP signaling and adaptation domain family and identified features that appear to be critical for receptor structure and function. Based on domain length and sequence conservation, we identified seven major MCP classes and three distinct structural regions within the cytoplasmic domain: signaling, methylation, and flexible bundle subdomains. The flexible bundle subdomain, not previously recognized in MCPs, is a conserved element that appears to be important for signal transduction. Remarkably, the N- and C-terminal helical arms of the cytoplasmic domain maintain symmetry in length and register despite dramatic variation, from 24 to 64 7-aa heptads in overall domain length. Loss of symmetry is observed in some MCPs, where it is concomitant with specific changes in the sensory module. Each major MCP class has a distinct pattern of predicted methylation sites that is well supported by experimental data. Our findings indicate that signaling and adaptation functions within the MCP cytoplasmic domain are tightly coupled, and that their coevolution has contributed to the significant diversity in chemotaxis mechanisms among different organisms. chemotaxis | methyl-accepting chemotaxis protein | signal transduction

Electron transfer dissociation (ETD) is a recently introduced mass spectrometric technique that provides a more comprehensive coverage of peptide sequences and posttranslational modifications. Here, we evaluated the use of ETD for a global phosphoproteome analysis. In all, we identified a total of 1,435 phosphorylation sites from human embryonic kidney 293T cells, of which 1,141 ([approximately equal to] 80%) were not previously described. A detailed comparison of ETD and collision-induced dissociation (CID) modes showed that ETD identified 60% more phosphopeptides than CID, with an average of 40% more fragment ions that facilitated localization of phosphorylation sites. Although our data indicate that ETD is superior to CID for phosphorylation analysis, the two methods can be effectively combined in alternating ETD and CID modes for a more comprehensive analysis. Combining ETD and CID, from this single study, we were able to identify 80% of the known phosphorylation sites in >1,000 phosphorylated peptides analyzed. A hierarchical clustering of the identified phosphorylation sites allowed us to discover 15 phosphorylation motifs that have not been reported previously. Overall, ETD is an excellent method for localization of phosphorylation sites and should be an integral component of any strategy for comprehensive phosphorylation analysis. bioinformatics | motifs | phosphorylation | signal transduction | systems biology

Results of protein sequence comparison at open criterion show a very large number of relationships that have, up to now, gone unreported. The relationships suggest many ancient events of gene duplication. It is well known that gene duplication has been a major process in the evolution of genomes. A collection of human genes that have known functions have been examined for a history of gene duplications detected by means of amino acid sequence similarity by using BLASTp with an expectation of two or less (open criterion). Because the collection of genes in build 35 includes sets of transcript variants, all genes of known function were collected, and only the longest transcription variant was included, yielding a 13,298-member library called KGMV (for known genes maximum variant). When all lengths of matches are accepted, >97% of human genes show significant matches to each other. Many form matches with a large number of other different proteins, showing that most genes are made up from parts of many others as a result of ancient events of duplication. To support the use of the open criterion, all of the members of the KGMV library were twice replaced with random protein sequences of the same length and average composition, and all were compared with each other with BLASTp at expectation two or less. The set of matches averaged 0.35% of that observed for the KGMV set of proteins. open criterion | protein | relationships | sequence

We have studied patterns of DNA sequence variation and evolution for 22 genes located on the neo-X and neo-Y chromosomes of Drosophila miranda. As found previously, nucleotide site diversity is greatly reduced on the neo-Y chromosome, with a severely distorted frequency specutrum. There is also an accelerated rate of amino-acid sequence evolution on the neo-Y chromosome. Comparisons of nonsynonymous and silent variation and divergence suggest that amino-acid sequences on the neo-X chromosome are subject to purifying selection, whereas this is much weaker on the neo-Y. The same applies to synonymous variants affecting codon usage. There is also an indication of a recent relaxation of selection on synonymous mutations for genes on other chromosomes. Genes that are weakly expressed on the neo-Y chromosome appear to have a faster rate of accumulation of both nonsynonymous and unpreferred synonymous mutations than genes with high levels of expression, although the rate of accumulation when both types of mutation are pooled is higher for the neo-Y chromosome than for the neo-X chromosome even for highly expressed genes.

Gelatinase biosynthesis-activating pheromone (GBAP) is an autoinducing peptide involved in Enterococcus faecalis fsr quorum sensing, and its 11-amino-acid sequence has been identified in the C-terminal region of the 242-residue deduced fsrB product (J. Nakayama et al., Mol. Microbiol. 41:145-154, 2001). In this study, however, we demonstrated the existence of fsrD, encoding the GBAP propeptide, which is in frame with fsrB but is translated independently of fsrB. It was also demonstrated that FsrB', an FsrD segment-truncated FsrB, functions as a cysteine protease-like processing enzyme to generate GBAP from FsrD. This revised model is consistent with the staphylococcal agr system.

Autotrophic members of the Sulfolobales (Crenarchaeota) contain acetyl-coenzyme A (CoA)/propionyl-CoA carboxylase as the C[O.sub.2] fixation enzyme and use a modified 3-hydroxypropionate cycle to assimilate C[O.sub.2] into cell material. In this central metabolic pathway malonyl-CoA, the product of acetyl-CoA carboxylation, is further reduced to 3-hydroxypropionate. Extracts of Metallosphaera sedula contained NADPH-specific malonyl-CoA reductase activity that was 10-fold up-regulated under autotrophic growth conditions. Malonyl-CoA reductase was partially purified and studied. Based on N-terminal amino acid sequencing the corresponding gene was identified in the genome of the closely related crenarchaeum Sulfolobus tokodaii. The Sulfolobus gene was cloned and heterologously expressed in Escherichia coli, and the recombinant protein was purified and studied. The enzyme catalyzes the following reaction: malonyl-CoA + NADPH + [H.sup.+] [right arrow] malonate-semialdehyde + CoA + [NADP.sup.+]. In its native state it is associated with small RNA. Its activity was stimulated by [Mg.sup.2+] and thiols and inactivated by thiol-blocking agents, suggesting the existence of a cysteine adduct in the course of the catalytic cycle. The enzyme was specific for NADPH ([K.sub.m] = 25 [micro]M) and malonyl-CoA ([K.sub.m] = 40 [micro]M). Malonyl-CoA reductase has 38% amino acid sequence identity to aspartate-semialdehyde dehydrogenase, suggesting a common ancestor for both proteins. It does not exhibit any significant similarity with malonyl-CoA reductase from Chloroflexus aurantiacus. This shows that the autotrophic pathway in Chloroflexus and Sulfolobaceae has evolved convergently and that these taxonomic groups have recruited different genes to bring about similar metabolic processes.

Cholesterol-dependent cytolysins (CDCs) are secreted, pore-forming toxins that are associated with pathogenesis in a variety of gram-positive bacteria. Bacillus anthracis produces anthrolysin O (ALO), a CDC that is largely responsible for the hemolytic activity of culture supernates when the bacterium is cultured in appropriate conditions. B. cereus and B. thuringiensis, species closely related to B. anthracis, produce CDCs with significant amino acid sequence homology to ALO. Transcription of the B. cereus and B. thuringiensis CDC genes is controlled by PlcR, a transcription regulator that requires a pentapeptide derived from the papR gene product for binding to a consensus sequence (PlcR box) and transcriptional activation of downstream genes. A PlcR box precedes the B. anthracis alo gene, and the B. anthracis genome contains three plcR-like genes, one of which harbors a nonsense mutation that is predicted to result in a truncated, nonfunctional protein. We detected mRNA of alo, papR, and the three plcR-like genes in spleens of B. anthracis-infected mice, indicating gene expression in vivo. Analysis of alo transcription in batch culture revealed a potential transcription start located between the PlcR box and the translational start. Nevertheless, steady-state levels of alo transcripts and ALO protein were unaffected by deletion of papR or disruption of the PlcR box. Our data indicate that despite the presence of the transcriptionally active plcR and papR genes in B. anthracis and a PlcR box in the promoter region of the alo gene, alo expression is independent of this control system.

Candida parapsilosis is responsible for severe cases of non-albicans systemic candidiasis and is one of the leading causes of mortality in neonates. The molecular mechanisms underlying this organism's virulence remain unknown. Unlike C. albicans, which can exist in several morphogenetic forms, C. parapsilosis exists in either the yeast or pseudohyphal forms. The environmental signals that trigger pseudohyphal differentiation and the signalling pathways that transduce these signals are unknown. This paper provides evidence for the role of amino acids in morphogenesis in C. parapsilosis. The cell and colony morphologies, pseudohyphal differentiation and invasive growth of five C. parapsilosis isolates were characterized in ammonium-rich minimal media lacking or supplemented with naturally occurring amino acids. C. parapsilosis underwent dramatic changes in cellular and colony morphology and formed pseudohyphae in response to a specific subset of amino acids. Transport studies showed that these amino acid inducers activate the transport of some, but not all, unrelated amino acids. Interestingly, citrulline, an amino acid that is not transported in the presence of ammonium, strongly induced pseudohyphal morphogenesis in C. parapsilosis under these conditions. Together the data suggest that amino acids are important morphogens in C. parapsilosis and that amino-acid-mediated morphogenesis in this organism does not require transport of the ligand across the plasma membrane.

The genomics revolution has unveiled a wealth of poorly characterized proteins. Scientists are often able to produce milligram quantities of proteins for which function is unknown or hypothetical, based only [...]

In protein structure space, protein structures cluster into four elongated regions when mapped based solely on similarity among the 3D structures. These four regions correspond to the four major classes of present-day proteins defined by the contents of secondary structure types and their topological arrangement. Evolution of and restriction to these four classes suggest that, in most cases, the evolution of genes may have been constrained or selected to those genetic changes that results in structurally stable proteins occupying one of the four 'allowed' regions of the protein structure space, 'structural selection,' an important component of natural selection in gene evolution. Our studies on tracing the 'common structural ancestor' for each protein sequence family of known structure suggest that: (i) recently emerged proteins belong mostly to three classes; (ii) the proteins that emerged earlier evolved to gain a new class; and (iii) the proteins that emerged earliest evolved to become the present-day proteins in the four major classes, with the fourth-class proteins becoming the most dominant population. Furthermore, our studies also show that not all present-day proteins evolved from one single set of proteins in the last common ancestral organism, but new common ancestral proteins were 'born' at different evolutionary times, not traceable to one or two ancestral proteins: 'the multiple birth model' for the evolution of protein sequence families. protein fold classes | common structural ancestor | evolutionary age | protein structure universe

The mass defect, that is, the difference between the nominal and actual monoisotopic masses, of a phosphorus in a phosphate group is greater than for most other atoms present in proteins. When the mass defects of tryptic peptides derived from the human proteome are plotted against their masses, phosphopeptides tend to fall off the regression line. By calculating the masses of all potential tryptic peptides from the human proteome, we show that regions of higher phosphorylation probability exist on such a plot. We developed a transformation function to estimate the mass defect of a peptide from its monoisotopic mass and empirically defined a simple formula for a user-selectable discriminant line that categorizes a peptide mass according to its probability of being phosphorylated. Our method performs similarly well on phosphopeptides derived from a database of experimentally validated phosphoproteins. The method is relatively insensitive to mass measurement error of up to 20 ppm. The approach can be used with a tandem mass spectrometer in real time to rapidly select and rank order the possible phosphopeptides from a mixture of unmodified peptides for subsequent phosphorylation site mapping and peptide sequence analysis.

The crystal structures of a single coiled-coil peptide in distinct parallel and antiparallel tetrameric configurations and parallel or antiparallel crystal structures of several related peptide sequences are analyzed and described. Results reveal that substitution of a single solvent-exposed residue enabled the parallel coiled-coli tetramer GCN4-pLI to populate the antiparallel configuration, suggesting that the two configurations are close enough in energy for the subtle sequence changes to have important structural consequences.

Recent data support the hypothesis that reactive oxygen species (ROS) play a central role in the initiation and progression of vascular diseases. An important vasoprotective function related to the regulation of ROS levels appears to be the antioxidant capacity of nitric oxide (NO). We previously reported that treatment with NO decreases phosphotyrosine levels of adapter protein [p130.sup.cas] by increasing protein tyrosine phosphatase-proline, glutamate, serine, and threonine sequence protein (PTP-PEST) activity, which leads to the suppression of agonist-induced [H.sub.2][O.sub.2] elevation and motility in cultured rat aortic smooth muscle cells (SMCs). The present study was performed to investigate the hypotheses that 1) IGF-I increases the activity of the small GTPase Rac1 as well as H202 levels and 2) NO suppresses IGF-I-induced [H.sub.2][O.sub.2] elevation by decreasing Rac1 activity via increased PTP-PEST activity and dephosphorylation of [p130.sup.cas]. We report that IGF-I induces phosphorylation of [p130.sup.cas] and activation of Rac1 and that NO attenuates these effects. The effects of NO are mimicked by the overexpression of PTP-PEST or dominant-negative (dn)-[p130.sup.cas] and antagonized by the expression of dn-PTP-PEST or [p130.sup.cas]. We conclude that IGF-I induces rat aortic SMC motility by increasing phosphotyrosine levels of [p130.sup.cas] and activating Rac1 and that NO decreases motility by activating PTP-PEST, inducing dephosphorylating [p130.sup.cas], and decreasing Rac1 activity. Decreased Rac1 activity lowers intracellular [H.sub.2][O.sub.2] levels, thus attenuating cell motility. hydrogen peroxide; protein tyrosine phosphatase-proline, glutamate, serine, and threonine sequence protein; [p130.sup.cas]

Synthetic peptides corresponding to the amino-terminal sequence of the beta chain of fibrin increase the turbidity of fibrin clots, whether they are generated by the direct interaction of thrombin and fibrinogen or by the reassociation of fibrin monomers. The comparable but delayed interaction involving the B knob and the betaC hole is ultimately directed at preparing the clot for its eventual destruction, although the interaction involving the A knob and gammaC is the basis for the polymerization of fibrin.

Correct identification of a peptide sequence from MS/MS data is still a challenging research problem, particularly in proteomic analyses of higher eukaryotes where protein databases are large. The scoring methods of search programs often generate cases where incorrect peptide sequences score higher than correct peptide sequences (referred to as distraction). Because smaller databases yield less distraction and better discrimination between correct and incorrect assignments, we developed a method for editing a peptide-centric database (PC-DB) to remove unlikely sequences and strategies for enabling search programs to utilize this peptide database. Rules for unlikely missed cleavage and nontryptic proteolysis products were identified by data mining 11 849 high-confidence peptide assignments. We also evaluated ion exchange chromatographic behavior as an editing criterion to generate subset databases. When used to search a well-annotated test data set of MS/MS spectra, we found no loss of critical information using PC-DBs, validating the methods for generating and searching against the databases. On the other hand, improved confidence in peptide assignments was achieved for tryptic peptides, measured by changes in ACN and RSP. Decreased distraction was also achieved, consistent with the 3-9-fold decrease in database size. Data mining identified a major class of common nonspecific proteolytic products corresponding to leucine aminopeptidase (LAP) cleavages. Large improvements in identifying LAP products were achieved using the PC-DB approach when compared with conventional searches against protein databases. These results demonstrate that peptide properties can be used to reduce database size, yielding improved accuracy and information capture due to reduced distraction, but with little loss of information compared to conventional protein database searches.

Identification of Salmonella serotypes is based on flagellar and somatic antigens. The absence of flagella may consequently affect complete identification of the serotype; here it is shown that Salmonella enterica serovar Typhimurium exhibits morphological differences dependent on the peptone constituents of the culture medium. Aflagellate salmonella were produced in certain media where the nutritional ingredient was casein-based peptone or gelatin-based peptone; in gelatin-based peptone, aggregates of salmonella were observed. However, in media containing soy-based peptone as the primary nutrient, salmonella displayed a normal flagellated morphology. Transfer of aflagellate salmonella from nutritionally poor media, with casein- or gelatin-based peptone, into rich nutrient broth allowed flagella synthesis, indicating that the aflagellate form is still able to produce flagella. Amino acid sequencing of the peptones producing aflagellate organisms showed a relatively low tyrosine concentration: only 0.03 [+ or -] 0.01 g [l.sup.-1] for gelatin-based buffered peptone water, compared to 0.21 [+ or -] 0.01 for soy-based buffered peptone water. Tyrosine is essential for flagellin, which is the subunit of the salmonella flagellar filament. The addition of 200 [micro]M tyrosine to casein-based peptone media produced flagellate salmonella; 2 mM glucose was needed in addition to tyrosine to achieve a similar morphology in gelatin-based media. Therefore, culture media containing less than 1.20 g tyrosine [l.sup.-1], and of limited carbohydrate source, when used for serological testing of clinical isolates, may result in an incomplete serological identification.

An essential component of the immune system of animals is the production of antimicrobial peptides (AMPs). In vertebrates and termites the protein sequence of some AMPs evolves rapidly under positive selection, suggesting that they may be coevolving with pathogens. However, antibacterial peptides in Drosophila tend to be highly conserved. We have inferred the selection pressures acting on Drosophila antifungal peptides (drosomycins) from both the divergence of drosomycin genes within and between five species of Drosophila and polymorphism data from Drosophila simulans and D. melanogaster. In common with Drosophila antibacterial peptides, there is no evidence of adaptive protein evolution in any of the drosomycin genes, suggesting that they do not coevolve with pathogens. It is possible that this reflects a lack of specific fungal and bacterial parasites in Drosophila populations. The polymorphism data from both species differed from neutrality at one locus, but this was not associated with changes in the protein sequence. The synonymous site diversity was greater in D. simulans than in D. melanogaster, but the diversity both upstream of the genes and at nonsynonymous sites was similar. This can be explained if both upstream and nonsynonymous mutations are slightly deleterious and are removed more effectively from D. simulans due to its larger effective population size.

Algorithmic search engines bridge the gap between large tandem mass spectrometry data sets and the identification of proteins associated with biological samples. Improvements in these tools can greatly enhance biological discovery. We present a new scoring scheme for comparing tandem mass spectra with a protein sequence database. The MASPIC (Multinomial Algorithm for Spectral Profile-based Intensity Comparison) scorer converts an experimental tandem mass spectrum into a m/z profile of probability and then scores peak lists from potential candidate peptides using a multinomial distribution model. The MASPIC scoring scheme incorporates intensity, spectral peak density variations, and m/z error distribution associated with peak matches into a multinomial distribution. The scoring scheme was validated on two standard protein mixtures and an additional set of spectra collected on a complex ribosomal protein mixture from Rhodopseudomonas palustris. The results indicate a 5-15% improvement over Sequest for high-confidence identifications. The performance gap grows as sequence database size increases. Additional tests on spectra from proteinase-K digest data showed similar performance improvements demonstrating the advantages in using MASPIC for studying proteins digested with less specific proteases. All these investigations show MASPIC to be a versatile and reliable system for peptide tandem mass spectral identification.

The Tol-Pal proteins of the cell envelope of Escherichia coli are required for maintaining outer membrane integrity. This system forms protein complexes in which TolA plays a central role by providing a bridge between the inner and outer membranes via its interaction with the Pal lipoprotein. The Tol proteins are parasitized by filamentous bacteriophages and group A colicins. The N-terminal domain of the Ff phage g3p protein and the translocation domains of colicins interact directly with TolA during the processes of import through the cell envelope. Recently, a four-amino-acid sequence in Pal has been shown to be involved in Pal's interaction with TolA. A similar motif is also present in the sequence of two TolA partners, g3p and colicin A. Here, a mutational study was conducted to define the function of these motifs in the binding activity and import process of TolA. The various domains were produced and exported to the bacterial periplasm, and their cellular effects were analyzed. Cells producing the g3p domain were tolerant to colicins and filamentous phages and had destabilized outer membranes, while g3p deleted of three residues in the motif was affected in TolA binding and had no effect on cell integrity or colicin or phage import. A conserved Tyr residue in the colicin A translocation domain was involved in TolA binding and colicin A import. Furthermore, in vivo and in vitro coprecipitation analyses demonstrated that colicin A and g3p N-terminal domains compete for binding to TolA.

A novel intracellular poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZd) of Wautersia eutropha (formerly Ralstonia eutropha) H16 which shows similarity with the catalytic domain of the extracellular PHB depolymerase in Ralstonia pickettii T1 was identified. The positions of the catalytic triad ([Ser.sup.190]-[Asp.sup.266]-[His.sup.330]) and oxyanion hole ([His.sup.108]) in the amino acid sequence of PhaZd deduced from the nucleotide sequence roughly accorded with those of the extracellular PHB depolymerase of R. pickettii T2, but a signal peptide, a linker domain, and a substrate binding domain were missing. The PhaZd gene was chined and the gene product was purified from Escherichia coli. The specific activity of PhaZd toward artificial amorphous PHB granules was significantly greater than that of other known intracellular PHB depolymerase or 3-hydroxybutyrate (3HB) oligomer hydrolases of IV. eutropha H16. The enzyme degraded artificial amorphous PHB granules and mainly released various 3-hydroxybutyrate olignmers. PhaZd distributed nearly equally between PHB inclusion bodies and the cytosolic fraction. The amount of PHB was greater in phaZd deletion mutant cells than the wild-type cells under various culture conditions. These results indicate that PhaZd is a novel intracelhdar PHB depolymerase which participates in the mobilization of PHB in W. eutropha H16 along with other PHB depolymerases.

Infrared spectroscopy was used to study the secondary structure of peptides, which imitate the amino acid sequences of the C-terminal domains of the pro-apoptotic Bak, and anti-apoptotic Bcl-2 proteins when they are inserted in lipid vesicles. It was concluded that the domains only form transmembrane helices in membranes of reduced thickness and that hydrophobic mismatching occurs in thicker membranes.

The BceB protein of the cystic fibrosis mucoid isolate Burkholderia cenocepacia IST432 is proposed to catalyze the first step of the exopolysaccharide repeat unit assembly. Extracts of Escherichia coli cells overexpressing BceB were shown to contain glycosyltransferase activity and mediate incorporation of glucose-l-phosphate into membrane lipids. The amino acid sequence of BceB exhibits two conserved regions, one comprising two invariant aspartic acid residues (Asp339 and Asp355) that are essential for catalysis, as substantiated by site-directed mutagenesis, and the other comprising a putative Rossmann fold motif. The results of protein topology analysis using PhoA and LacZ fusions supported in silico predictions that BceB has at least six transmembrane segments and two major cytoplasmic loops comprising the conserved regions described above.

A citrate synthase (CS) deletion mutant of Agrobacterium tumefaciens C58 is highly attenuated in virulence. The identity of the mutant was initially determined from its amino acid sequence, which is 68% identical to Escherichia coli and 77% identical to Brueella melitensis. The mutant lost all CS enzymatic activity, and a cloned CS gene complemented a CS mutation in Sinorhizobium. The CS mutation resulted in a 10-fold reduction in vir gene expression, which likely accounts for the attenuated virulence. When a plasmid containing a constitutive virG [virG(Con)] locus was introduced into this mutant, the level of vir gene induction was restored to nearly wild-type level. Further, the virG(Con)-complemented CS mutant strain induced tumors that were similar in size and number to those induced by the parental strain. The CS mutation resulted in only a minor reduction in growth rate in a glucose-salts medium. Both the CS mutant and the virG(Con)-complemented CS strain displayed similar growth deficiencies in a glucose-salts medium, indicating that the reduced growth rate of the CS mutant could not be responsible for the attenuated virulence. A search of the genome of A. tumefaciens C58 revealed four proteins, encoded on different replicons, with conserved CS motifs. However, only the locus that when mutated resulted in an attenuated phenotype has CS activity. Mutations in the other three loci did not result in attenuated virulence and any loss of CS activity, and none were able to complement the CS mutation in Sinorhizobium. The function of these loci remains unknown.

The effect of the E170K and Q252L mutations on the secondary and quaternary structures of glucose dehydrogenase (GlcDH) was examined. The results indicated that the stability of the Q252L/E170K GlcDH is driven by stabilization of the quaternary structure and that a hydrophobic cavity is formed at the subunit-subunit interfaces.

The phoU gene of Aquifex aeolicus encodes a protein called PHOU_AQUAE with sequence similarity to the PhoU protein of Escherichia coil Despite the fact that there is a large number of family members (more than 300) attributed to almost all known bacteria and despite PHOU_AQUAE's association with the regulation of genes for phosphate metabolism, the nature of its regulatory, function is not well understood. Nearly one-half of these PhoU-like proteins, including both PHOU_AQUAE and the one from E. coli, form a subfamily with an apparent dimer structure of two PhoU domains on the basis of their amino acid sequence. The crystal structure of PHOU_AQUAE (a 221-amino-acid protein) reveals two similar coiled-coil PhoU domains, each forming a three-helix bundle. The structures of PHOU_AQUAE proteins from both a soluble fraction and refolded inclusion bodies (at resolutions of 2.8 and 3.2 [Angstrom], respectively) showed no significant differences. The folds of the PhoU domain and Bag domains (for a class of cofactors of the eukaryotic chaperone Hsp70 family) are similar. Accordingly, we propose that gene regulation by PhoU may occur by association of PHOU_AQUAE with the ATPase domain of the histidine kinase PhoR, promoting release of its substrate PhoB. Other proteins that share the PhoU domain fold include the coiled-coil domains of the STAT protein, the ribosome-recycling factor, and structural proteins like spectrin.