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Author(s): Nicole A. Doria-Rose [sup.1] , Chaim A. Schramm [sup.2] , Jason Gorman [sup.1] , Penny L. Moore [sup.3] [sup.4] [sup.5] , Jinal N. Bhiman [sup.3] [sup.4] , Brandon J. [...], Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development. A longitudinal study of an individual patient developing neutralizing antibodies against HIV-1 (targeting the V1V2 region of gp120) reveals how such neutralizing antibodies develop and evolve over time, providing important insights relevant to vaccine development. HIV-neutralizing antibody formation examined A better understanding of how HIV-1-neutralizing antibodies are generated could be a useful contribution to the design of improved AIDS vaccines. John Mascola and colleagues have now elucidated the immunological pathway of an important category of HIV-1-neutralizing antibody -- those that target the variable V1V2 region of the viral envelope. These antibodies are more frequently elicited than CD4-binding site antibodies in the early stages of HIV infection and feature modest affinity maturation, a process that favours mutations in antibody variable domains that enhance antigen binding.

The emergence of multi-drug-resistant strains of Staphylococcus epidermidis emphasizes the need to develop new antibiotics. The unique and essential role of the peptide deformylase (PDF) in catalysing the removal of the N-terminal formyl group from newly synthesized polypeptides in eubacteria makes it an attractive antibacterial drug target. In the present study, both deformylase homologues from S. epidermidis (SePDF-1 and SePDF-2) were cloned and expressed, and their enzymic activities were characterized. [Co.sup.2+]-substituted SePDF-1 exhibited much higher enzymic activity ([k.sub.cat]/[K.sub.m] 6.3 x [10.sup.4] [M.sup.-1] [s.sup.-1]) than those of [Ni.sup.2+]-and [Zn.sup.2+]- substituted SePDF-1, and SePDF-1 showed much weaker binding ability towards [Ni.sup.2+] than towards [Co.sup.2+] and [Zn.sup.2+], which is different from PDF in Staphylococcus aureus (SaPDF), although they share 80% amino-acid sequence identity. The determined crystal structure of SePDF-1 was similar to that of (SaPDF), except for differences in the metal-binding sites. The other deformylase homologue, SePDF-2, was shown to have no peptide deformylase activity; the function of SePDF-2 needs to be further investigated. DOI 10.1099/mic.0.038174-0

Computational studies of the relationships between protein sequence, structure, and folding have traditionally relied on purely local sequence representations. Here we show that global representations, on the basis of parameters that encode information about complete sequences, contain otherwise inaccessible information about the organization of sequences. By studying the spectral properties of these parameters, we demonstrate that amino acid physical properties fall into two distinct classes. One class is comprised of properties that favor sequentially localized interaction clusters. The other class is comprised of properties that favor globally distributed interactions. This observation provides a bridge between two classic models of protein folding--the collapse model and the nucleation model--and provides a basis for understanding how any degree of intermediacy between these two extremes can occur. proteomics | sequence analysis doi/10.1073/pnas.1001299107

A competitive ATP-dependent mechanism is found to be involved in the inhibition of Salmonella enterica serovar Typhimurium Lon protease by the peptidyl boronates, based on the amino acid sequence of a product of peptide hydrolysis by the enzyme. The peptidyl boronate is found to be extremely beneficial and useful for future studies, as it contains a fluorescent dansyl moiety with which the various interactions can be monitored easily.

Physics-based analytical forms are proposed to express the cavity component of the free energy of hydrophobic association of spherical and spheroidal solute particles. These expressions are derived on the basis of an analysis of the change of the number and free energy of water molecules upon the formation of a hydrophobic dimmer and a Gaussian model of the density of the solute.

Previous work has shown that the C-terminal portion of BphA, especially two amino acid segments designated region III and region IV, influence the regiospecificity of the biphenyl dioxygenase (BPDO) toward 2,2'-dichlorobiphenyl (2,2'-CB). In this work, we evolved BPDO by shuffling bphA genes amplified from polychlorinated biphenyl-contaminated soil DNA. Sets of approximately 1-kb DNA fragments were amplified with degenerate primers designed to amplify the C-terminal portion of bphA. These fragments were shuffled, and the resulting library was used to replace the corresponding fragment of Burkholderia xenovorans LB400 bphA. Variants were screened for their ability to oxygenate 2,2'-CB onto carbons 5 and 6, which are positions that LB400 BPDO is unable to attack. Variants S100, S149, and S151 were obtained and exhibited this feature. Variant S100 BPDO produced exclusively cis-5,6-dihydro-5,6-dihydroxy-2,2'-dichlorobiphenyl from 2,2'-CB. Moreover, unlike LB400 BPDO, S100 BphA catalyzed the oxygenation of 2,2',3,3'-tetrachlorobiphenyl onto carbons 5 and 6 exclusively and it was unable to oxygenate 2,2',5,5'-tetrachlorobiphenyl. Based on oxygen consumption measurements, variant S100 oxygenated 2,2'-CB at a rate of 16 [+ or -] 1 nmol [min.sup.-1] per nmol enzyme, which was similar to the value observed for LB400 BPDO. cis-5,6-Dihydro-5,6-dihydroxy-2,2'-dichlorobiphenyl was further oxidized by 2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB) and 2,3-dihydroxybiphenyl dioxygenase (BphC). Variant S100 was, in addition, able to oxygenate benzene, toluene, and ethyl benzene. Sequence analysis identified amino acid residues [M.sup.237] [S.sup.238] and [S.sup.283] outside regions III and IV that influence the activity toward doubly ortho-substituted chlorobiphenyls.

A study presents new information on alpha/beta-peptides that were designed to address questions about the relationships among alpha/beta-amino acid sequence, three-dimensional folding, physical properties, and biological activity. Findings support the conclusion that the global segregation of lipophilic and hydrophilic (cationic) side chains in a helical conformation is not essential for selective antibacterial activity.

Continued development of a new sequence--specific algorithm for peptide retention prediction in RP HPLC is reported. Our discovery of the large effect on the apparent hydrophobicity of N-terminal amino acids produced by the ion-pairing retention mechanism has led to the development of sequence-specific retention calculator (SSRCalc) algorithms. These were optimized for a set of ~2000 tryptic peptides confidently identified by off-line micro-HPLC-MALDI MS (MS/MS) (300-[Angstrom] pore size C18 sorbent, linear water/acetonitrile gradient, and trifluoroacetic acid as ion-pairing modifier). The latest version of the algorithm takes into account amino acid composition, position of the amino acid residues (N- and C-terminal), peptide length, overall hydrophobicity, pI, nearest-neighbor effect of charged side chains (K, R, H), and propensity to form helical structures. A correlation with [R.sup.2]~0.98 was obtained for the 2000-peptide optimization set. A flexible structure for the SSRC programming code allows easy adaptation to different chromatographic conditions. This was demonstrated by adapting the algorithm (~0.98 [R.sup.2] value) for a set of ~2500 peptides separated on a 100-[Angstrom] pore size C18 column. The SSRCalc algorithm has also been extensively tested for a number of real samples, providing solid support for protein identification and characterization; correlations in the range of 0.95-0.97 [R.sup.2] value have normally been observed.

A mutant of Vibrio vulnificus that was more sensitive to low pH was screened from a library of mutants constructed by random transposon mutagenesis. By use of a transposon-tagging method, an open reading frame encoding a LysR homologue, AphB, was identified and cloned from V. vulnificus. The deduced amino acid sequence of AphB from V. vulnificus was 80% identical to that reported from V. cholerae. A mutational analysis demonstrated that the gene product of aphB contributes to acid tolerance of V. vulnificus. The lysine decarboxylase activity and cellular level of the cadA transcript were decreased in the aphB mutant, indicating that AphB exerts its effect on the acid tolerance of V. vulnificus by enhancing the expression of cadBA. Western blot analyses demonstrated that the cellular level of CadC, a transcription activator of the cadBA operon, was significantly reduced by aphB mutation, and a primer extension analysis revealed that the cadC promoter ([P.sub.cadC]) activity was under the positive control of AphB. A direct interaction between AphB and the [P.sub.cadC] DNA was demonstrated by gel mobility shift assays. The AphB binding site mapped by deletion analyses of the [P.sub.cadC] regulatory region and confirmed by a DNase I protection assay was centered at the 61.5 bp upstream of the transcription start site. Accordingly, these results demonstrate that AphB and CadC function sequentially in a regulatory cascade to activate cadBA expression and that AphB activates the expression of cadC by directly binding to an upstream region of [P.sub.cadC].

We report here the application of electrospray ionization tandem mass spectrometry for the characterization of protein ubiquitylation, an important posttranslational modification of cellular proteins. Trypsin digestion of ubiquitin-conjugated proteins produces diglycine branched peptides containing the modification sites. Chemical derivatization by N-terminal sulfonation was carded out on several model peptides for the formation of a characteristic fragmentation pattern in their MS/MS analysis. The fragmentation of derivatized singly charged peptides results in a product ion distribution similar to that already observed by MALDI-TOF MS/MS. Signature fragments distinguished the diglycine branched peptides from other modified and unmodified peptides, while the sequencing product ions reveal the amino acid sequence and the location of the ubiquitylation site. Doubly charged peptide derivatives fragment in a somewhat different manner, but several fragments characteristic to diglycine branched peptides were observed under low collision energy conditions. These signature peaks can also be used to identify peptides containing ubiquitylation sites. In addition, a marker ion corresponding to a glycine-modified lysine residue produced by high-energy fragmentation provides useful information for identity verification. The method is demonstrated by the analysis of three ubiquitinconjugated proteins using IC/MS/MS.

There are a variety of reasons to reconstruct the sequences of ancient proteins, but whatever the reason, the value of the reconstructed protein depends on the accuracy with which the ancient sequence is inferred. This study uses sequences simulated by a sequence-evolution simulation program that compares parsimony, maximum likelihood, and the Bayesian methods of inferring ancestral sequences and concludes that the Bayesian method, as implemented by MRBAYES 3.11, is preferred. Estimated ancestral sequences are of necessity the same length as the alignment on which the underlying phylogeny is based. A highly accurate method for correcting the estimated sequences is introduced, and it is shown that the correction permits inferring the sequences of ancient protein sequences with a very high degree of accuracy. ancestral state | Bayesian | phylogenetics

Inspection of the genomic DNA sequence of the oral anaerobe Porphyromonas gingivalis reveals that the micro-organism possesses the peroxide-sensing transcription activator OxyR, but not the superoxide-sensing transcription factor SoxR. Investigatation of oxidative-stress-responsive proteins in P. gingivalis by two-dimensional gel electrophoresis showed that two proteins were predominantly upregulated in oxidative conditions. In a P. gingivalis oxyR mutant these two proteins were not induced by treatment with hydrogen peroxide under aerobic conditions. By N-terminal amino acid sequencing, the two proteins were found to be superoxide dismutase and alkyl hydroperoxide reductase, encoded by sod and ahpC, respectively. Northern blot and lacZ fusion analyses revealed that P. gingivalis sod and ahpC were positively regulated by OxyR. Primer extension analysis located the promoter regions of sod and ahpC, and putative -35 boxes of these promoters were found immediately adjacent to their putative OxyR-binding sequences. Moreover, the promoter regions of sod and ahpC had the ability to bind P. gingivalis OxyR protein. These results demonstrate that P. gingivalis sod is one of the OxyR regulons, suggesting that OxyR functions as an intracellular redox sensor rather than a peroxide sensor in this organism. A sodgene of Bacteroides fragilis, which is taxonomically related to P. gingivalis, is inducible by redox stresses but not controlled by its OxyR. A DNA fragment including the B. fragilis sod promoter region could bind the P. gingivalis OxyR protein; however, a putative OxyR binding sequence within the DNA fragment was 14 bases distant from a putative -35 box of its promoter.

The pathway for the synthesis of the organic solute glucosylglycerate (GG) is proposed based on the activities of the recombinant glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from Methanococcoides burtonii. A mannosyl-3-phosphoglycerate phosphatase gene homologue (mpgP) was found in the genome of M. burtonii (http://www.jgi.doe.gov), but an mpgS gene coding for mannosyl-3-phosphoglycerate synthase (MpgS) was absent. The gene upstream of the mpgP homologue encoded a putative glucosyltransferase that was expressed in Escherichia coli. The recombinant product had GpgS activity, catalyzing the synthesis of glucosyl-3-phosphoglycerate (GPG) from GDP-glucose and D-3-phosphoglycerate, with a high substrate specificity. The recombinant MpgP protein dephosphorylated GPG to GG and was also able to dephosphorylate mannosyl-3-phosphoglycerate (MPG) but no other substrate tested. Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from Thermus thermophilus, Pyrococcus horikoshii, and 'Dehalococcoides ethenogenes.' GpgS had maximal activity at 50[degrees]C. The maximal activity of GpgP was at 50[degrees]C with GPG as the substrate and at 60[degrees]C with MPG. Despite the similarity of the sugar donors GDP-glucose and GDP-mannose, the enzymes for the synthesis of GPG or MPG share no amino acid sequence identity, save for short motifs. However, the hydrolysis of GPG and MPG is carried out by phosphatases encoded by homologous genes and capable of using both substrates. To our knowledge, this is the first report of the elucidation of a biosynthetic pathway for glucosylglycerate.

The studies on different insulators from different organisms which identified common themes in their mode of action are presented. The result suggests that attachment of chromatin to the pore might be dynamic and an intrinsic aspect of gene activation helping pore proteins to function at boundaries to recruit chromatin remodeling and modify proteins to allow them to block the spread of silenced chromatin.

The Rhodobacter sphaeroides response regulator PrrA directly activates transcription of genes necessary for energy conservation at low [O.sub.2] tensions and under anaerobic conditions. It is proposed that PrrA homologues contain a C-terminal DNA-binding domain (PrrA-CTD) that lacks significant amino acid sequence similarity to those found in other response regulators. To test this hypothesis, single amino acid substitutions were created at 12 residues in the PrrA-CTD. These mutant PrrA proteins were purified and tested for the ability to be phosphorylated by the low-molecular-mass phosphate donor acetyl phosphate, to activate transcription and to bind promoter DNA. Each mutant PrrA protein accepted phosphate from [sup.32]P- labelled acetyl phosphate. At micromolar concentrations of acetyl phosphate-treated wild-type PrrA, a single 20 bp region in the PrrA-dependent cycA P2 promoter was protected from DNase I digestion. Of the mutant PrrA proteins tested, only acetyl phosphate-treated PrrA-N168A and PrrA-I177A protected cycA P2 from DNase I digestion at similar protein concentrations compared to wild-type PrrA. The use of in vitro transcription assays with the PrrA-dependent cycA P2 and puc promoters showed that acetyl phosphate-treated PrrA-N168A produced transcript levels similar to that of wild-type PrrA at comparable protein concentrations. Using concentrations of acetyl phosphate-treated PrrA that are saturating for the wild-type protein, PrrA-H170A and PrrA-I177A produced < 45 % as much transcript as wild-type PrrA. Under identical conditions, the remaining mutant PrrA proteins produced little or no detectable transcripts from either promoter in vitro. Explanations are presented for why these amino acid side chains in the PrrA-CTD are important for its ability to activate transcription.

The LA repeats that comprise the ligand-binding domain of the LDL receptor are among the most common autonomously structured extracellular models found in the non-redundant protein sequence database. The information content of the amino acid sequence of a typical LA module is investigated by constructing sequences with alanine residues at nonconserved positions in the module.

Mass spectrometry-based de novo peptide sequencing is generally more reliable on high-resolution instruments owing to their high resolution and mass accuracy. On a lower resolution instrument such as the more widely used quadrupole ion traps, de novo peptide sequencing is not so reliable or requires more M[S.sup.3] experiments. However, the peptide CID spectrum has been demonstrated to be quite reproducible on an ion trap instrument and can be predicted with good accuracy. A new de novo peptide sequencing technique, DACSIM, combining a divide-and-conquer algorithm for deriving sequence candidates and spectrum simulation for sequence refinement, is developed for spectra acquired on an ion trap instrument. When DACSIM was used to sequence peptides 500--1900 u in mass generated from proteolytic digests of hemoglobin and myoglobin, the success rate was 70% with a false positive rate of only 6%, when isoleucine and leucine residues were not distinguished.

Peptide sequence analysis using a combination of gas-phase ion/ion chemistry and tandem mass spectrometry (MS/MS) is demonstrated. Singly charged anthracene anions transfer an electron to multiply protonated peptides in a radio frequency quadrupole linear ion trap (QLT) and induce fragmentation of the peptide backbone along pathways that are analogous to those observed in electron capture dissociation. Modifications to the QLT that enable this ion/ion chemistry are presented, and automated acquisition of high-quality, single-scan electron transfer dissociation MS/MS spectra of phosphopeptides separated by nanoflow HPLC is described. electron capture dissociation | fragmentation | ion/ion reactions | charge transfer | ion trap

Results rveal that p-nitophenyl phosphatase, p34, bearing high sequence similarity to two yeast alkaline phosphatases, belongs to the group of phosphatases that exhibit sequence motof DXDX(T/V) as shown by BLAST search analysis. Metal ions enhance p34 activity and the parthenolide binding site of p34 is different from p34 active site.

A comparative analysis of the sequence of R7A symbiosis island with the putative strain MAFF303099 symbiosis island uncovers novel symbiotic genes, which have bearing on the evolution of the two mesorhizobium loti symbiosis islands. Data indicate that most of the 414 genes of the R7A island are strain-specific and clustered.

The amino acid sequence of the CAMP cohemolysin, Cam, from Riemerella anatipestifer exhibits high homology to those of O-sialoglycoprotein endopeptidases, suggesting that the cohemolysin is a sialoglycoprotease. The cohemolysin gene, cam, is confined to a 1026-basepair open reading frame and encodes a 37 kilodalton molecular size protein.

This article discusses the current status of the chemistry of catalysis of the biotin-dependent enzymes with reference to carboxylases, protein sequence, and structural information.

Research reveals an ambiguity in the identification of the amino acid residue 149 in Escherichia coli sigma(up)70. It is shown to be either arginine or aspartic acid, each of which is shown to influence the genetic manipulation of sigma(sup)70.

In this paper, an approach is described that combines multiple structure alignments and multiple sequence alignments to generate sequence profiles for protein families. First, multiple sequence alignments are generated from sequences that are closely related to each sequence of known three-dimensional structure. These alignments then are merged through a multiple structure alignment of family members of known structure. The merged alignment is used to generate a Hidden Markov Model for the family in question. The Hidden Markov Model can be used to search for new family members or to improve alignments for distantly related family members that already have been identified. Application of a profile generated for SH2 domains indicates that the Janus family of nonreceptor protein tyrosine kinases contains SH2 domains. This conclusion is strongly supported by the results of secondary structure-prediction programs, threading calculations, and the analysis of comparative models generated for these domains. One of the Janus kinases, human TYK2, has an SH2 domain that contains a histidine instead of the conserved arginine at the key phosphotyrosine-binding position, [beta]B5. Calculations of the p[K.sub.a] values of the [beta]B5 arginines in a number of SH2 domains and of the [beta]B5 histidine in a homology model of TYK2 suggest that this histidine is likely to be neutral around pH 7, thus indicating that it may have lost the ability to bind phosphotyrosine. If this indeed is the case, TYK2 may contain a domain with an SH2 fold that has a modified binding specificity.

We purified microtubules from a mammalian mitotic extract and obtained an amino acid sequence from each microtubule-associated protein by using mass spectrometry. Most of these proteins are known spindle-associated components with essential functional roles in spindle organization. We generated antibodies against a protein identified in this collection and refer to it as astrin because of its association with astral microtubule arrays assembled in vitro. Astrin is [approximately equal to] 134 kDa, and except for a large predicted coiled-coil domain in its C-terminal region it lacks any known functional motifs. Astrin associates with spindle microtubules as early as prophase where it concentrates at spindle poles. It localizes throughout the spindle in metaphase and anaphase and associates with midzone microtubules in anaphase and telophase. Astrin also localizes to kinetochores but only on those chromosomes that have congressed. Deletion analysis indicates that astrin's primary spindle-targeting domain is at the C terminus, although a secondary domain in the N terminus can target some of the protein to spindle poles. Thus, we have generated a comprehensive list of major mitotic microtubule-associated proteins, among which is astrin, a nonmotor spindle protein.

The random peptide library display technique reveals that Escherichia coli flagellin protein, in addition to the known proliferating cell nuclear antigen (PCNA)-binding motif, contains a second larger group of peptides consisting of different consensus sequence for PCNA-binding.

The sequence analysis of the elapid cobra venom metalloproteinase kaouthiagin shows a P-III type domain structure and an extra disintegrin-like sequence domain. Data point out that the domain is Cys-rich and is inhibitory to the agonist-induced platelet aggregation.

Results demonstrate that lysophospholipase A isolated from Legionella pneumophila cleaves lysophosphatidylcholine. The amino-terminal catalytic domain exhibits amino acid sequence composed of FGDSLS, similar to lipolytic enzymes.