Identification and analysis of novel amino acid sequence repeats and domains in Pyrobaculum aerophilum using computational tools

We have identified four repeats and five domains that are novel in proteins encoded by the Pyrobaculum aerophilum str. IM2 proteome using automated in silico methods. A "repeat" corresponds to a region comprising less than 55 amino acid residues that occurs more than once in the protein sequence and sometimes present in tandem. A "domain" corresponds to a conserved region comprising greater than 55 amino acid residues and may be present as single or multiple copies in the protein sequence. These correspond to (1) 85 amino acid residues AAG domain, (2) 72 amino acid residues GFGN domain, (3) 43 amino acid residues KGG repeat, (4) 25 amino acid residues RWE repeat, (5) 25 amino acid residues RID repeat, (6) 108 amino acid residues NDFA domain, (7) 140 amino acid residues VxY domain, (8) 35 amino acid residues LLPN repeat and (9) 98 amino acid residues GxY domain. A repeat or domain is characterized by specific conserved sequence motifs. We discuss the presence of these repeats and domains in proteins from other genomes and their probable secondary structure.     

Structure of the Bacillus brevis metalloprotease gene

Primary structure of DNA fragment of 2355 b.p., encoding metalloprotease gene of Bac. brevis, had been determined. Open reading frame for a protein with size of 528 amino acid residues was found in this sequence. The encoding protein is homologous to metalloproteases of Bac. stearothermophilus, Bac. cereus, Bac. subtilis and Bac. amyloliquefaciens. The structure of Bac. brevis metalloprotease gene reveals that this enzyme is synthesised as pre-pro-protease with signal peptide and pro-region, which are cut during its synthesis. The proposed size of mature protease is 304 amino acid residues. The residues, essential for catalysis, binding of Zn ion and Ca ions were found on the basis of Bacilli metalloproteases structures comparison.     

Mechanistic and functional studies of the interaction of a proline-rich antimicrobial peptide with mammalian cells

Mammalian antimicrobial peptides provide rapid defense against infection by inactivating pathogens and by influencing the functions of cells involved in defense responses. Although the direct antibacterial properties of these peptides have been widely characterized, their multiple effects on host cells are only beginning to surface. Here we investigated the mechanistic and functional aspects of the interaction of the proline-rich antimicrobial peptide Bac7(1-35) with mammalian cells, as compared with a truncated analog, Bac7(5-35), lacking four critical N-terminal residues (RRIR) of the Bac7(1-35) sequence. By using confocal microscopy and flow cytometry, we showed that although the truncated analog Bac7(5-35) remains on the cell surface, Bac7(1-35) is rapidly taken up into 3T3 and U937 cells through a nontoxic energy- and temperature-dependent process. Cell biology-based assays using selective endocytosis inhibitors and spectroscopic and surface plasmon resonance studies of the interaction of Bac7(1-35) with phosphatidylcholine/cholesterol model membranes collectively suggest the concurrent contribution of macropinocytosis and direct membrane translocation. Structural studies with model membranes indicated that membrane-bound Bac7(5-35) is significantly more aggregated than Bac7(1-35) due to the absence of the N-terminal cationic cluster, thus providing an explanation for hampered cellular internalization of the truncated form. Further investigations aimed to reveal functional implications of intracellular uptake of Bac7(1-35) demonstrated that it correlates with enhanced S phase entry of 3T3 cells, indicating a novel function for this proline-rich peptide.     

The primary structure of D-amino acid oxidase from pig kidney. I. Isolation and sequence of the tryptic peptides

D-Amino acid oxidase from pig kidney cortex was digested with trypsin. Thirty-two tryptic peptides were isolated by ion exchange chromatography, high voltage paper electrophoresis, descending paper chromatography, and reverse-phase high performance liquid chromatography. The last method permitted the isolation of 29 tryptic peptides, many in a single step, in yields usually greater than 75%. The purified peptides were characterized by amino acid analysis and their sequences determined by the manual 5-dimethylaminonaphthalene-1-sulfonyl-Edman degradation procedure or by the automated Edman-Begg degradation method. These peptides accounted for all 12 lysine and 21 arginine residues observed by amino acid analysis of the intact protein and for 347 amino acid residues of the 345 predicted by the analysis.     

Biochemical properties and hormonal regulation of barley nuclease

The amino acid composition and NH2-terminal amino acid sequence of barley nuclease (EC 3.1.30.2) were determined. The amino acid composition is similar to that of mung bean nuclease, and therefore the biochemical properties of barley nuclease were characterized and compared with those of mung bean and other plant nucleases. The 3'-nucleotidase activity of barley nuclease is greater for purine than for pyrimidine ribonucleotides. The enzyme has little activity towards ribonucleoside 2' and 5'-monophosphates, and deoxyribonucleoside 3' and 5'-monophosphates, and is also inactive towards the 3'-phosphoester linkage of nucleoside cyclic 2',3' and 3',5'-monophosphates. The enzyme hydrolyzes dinucleoside monophosphates, showing strong preference for purine nucleosides as the 5' residues. Barley nuclease shows significant base preference for homoribonucleic acids, catalyzing the hydrolysis of polycytidylic acid greater than polyuridylic acid greater than polyadenylic acid much greater than polyguanylic acid. The enzyme also has preference for single-stranded nucleic acids. Hydrolysis of nucleic acids is primarily endonucleolytic, whereas the products of digestion possess 5'-phosphomonoester groups. Nuclease activity is inhibited by ethylenediaminetetraacetic acid and zinc is required for reactivation. Secretion of nuclease from barley aleurone layers is dependent on the hormone gibberellic acid [Brown, P.H. and Ho, T.-h. D. (1986) Plant Physiol. 82, 801-806]. Consistent with these results, gibberellic acid induces up to an eight-fold increase in the de novo synthesis of nuclease in aleurone layers. The secreted enzyme is a glycoprotein having an apparent molecular mass of 35 kDa. It consists of a single polypeptide having an asparagine-linked, high-mannose oligosaccharide. The protein portion of the molecule has a molecular mass of 33 kDa.     

Molecular organization of the N-terminal region of delta-endotoxin of Bacillus thuringiensis subspecies alesti

The tryptic peptide sequences of the N-terminal domain ("true toxin") of delta-endotoxin of Bac. thuringiensis subspecies alesti carrying 282 amino acid residues were determined. A comparison of these sequences with the primary structures of delta-endotoxin of subspecies kurstaki (K-1, K-73) determined by an analysis of corresponding structural genes revealed a conservative region of "true toxin" (residues 29-346) and a hypervariable region (residues 347-617) carrying multiple (not less than 50%) substituents of amino acid residues. It is essential that the amino acid substituents in the variable region are distributed unevenly, being grouped into several highly variable sites carrying 7 to 31 residues. Besides, tryptic peptides of subspecies alesti delta-endotoxin were found to contain peptides having no homologs in the structures of subspecies kurstaki delta-endotoxins. It seems probable that such an uneven distribution of amino acid substituents in the structures of delta-endotoxins of subspecies alesti and kurstaki reflects the functional differences in the two halves of the N-terminal domain ("true toxin"), one of which (i. e., conservative) may be responsible for the toxic effect, while the other one (i. e., variable) seems to participate in toxin interactions with the appropriate receptors of larvae gut.     

Purification and partial characterization of a novel antibacterial agent (Bac1829) Produced by Staphylococcus aureus KSI1829.

A novel antimicrobial agent from Staphylococcus aureus KSI1829, designated Bac1829, was purified by sequential steps of ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and hydrophobic interaction chromatography. Purified Bac1829 has a molecular mass of 6,418 +/- 2 Da. The peptide in heat stable, since full biological activity is retained after heating at 95 degrees C for 15 min, and it is destroyed by digestion with proteases. Amino acid sequence analysis revealed a high concentration of Ala and Gly residues, which respectively comprised 24 and 19% of the total amino acid content. Additionally, high levels of hydrophobic amino acids were present, accounting for the hydrophobic nature of Bac1829. Purified Bac1829 killed exponentially growing Corynebacterium renale in a dose-dependent manner by a bactericidal mode of action. A partial inhibitory spectrum analysis revealed that the following organisms were sensitive to the inhibitory activity of Bac1829: S. aureus RN4220, Streptococcus suis, Corynebacterium pseudotuberculosis, C. renale, Corynebacterium diptheriae, Haemophilus parasuis, Bordetella pertussis, Bordetella bronchoseptica, Moraxella bovis, and Pasteurella multocida.     

Purification, characterization, and primary structure of a novel cell wall hydrolytic amidase, CwhA, from Achromobacter lyticus

A novel bacteriolytic enzyme CwhA (cell wall hydrolytic amidase) was purified by ion exchange and gel-filtration chromatographies from a commercial bacteriolytic preparation from Achromobacter lyticus. CwhA exhibited optimal pH at 8.5 and lysed CHCl(3)-treated Escherichia coli more efficiently than Micrococcus luteus, Staphylococcus aureus, Enterococcus faecalis, and Pediococcus acidilactici. The enzyme was inhibited by 1,10-phenanthroline strongly and by EDTA to a lesser extent, suggesting that it is probably a metalloenzyme. Amino acid composition and mass spectrometric analyses for the CwhA-derived M. luteus muropeptides revealed that CwhA is N-acetylmuramoyl-L-alanine amidase [EC 3.5.1. 28]. The complete amino acid sequence of CwhA was established by a combination of Edman degradation and mass spectrometry for peptides obtained by Achromobacter protease I (API) digestion and cyanogen bromide (CNBr) cleavage. The enzyme consists of a single polypeptide chain of 177 amino acid residues with one disulfide bond, Cys114-Cys121. CwhA was found to be homologous to N-acetylmuramoyl-L-alanine amidase from bacteriophage T7 (BPT7). Its sequence identity with BPT7 is 35%, but the amino acid residues functioning as zinc ligands in BPT7 are absent in CwhA. These results suggest that CwhA is a new type of N-acetylmuramoyl-L-alanine amidase.     

Isolation and characterization of hagfish thyroid iodoprotein by its non-thyroglobulin nature, very high iodine and carbohydrate contents and low hormone/iodine ratio

We have characterized the thyroid iodoprotein of a hagfish, Eptatretus burgeri, one of the lowest marine vertebrates. The iodoprotein was not very homogeneous in its apparent molecular mass which decreased with the increase in hormone/iodotyrosine ratio. Four subfractions with an apparent molecular mass of about 400 kDa were purified from one major fraction by size-exclusion and Mono Q ion-exchange HPLC. The subfractions appeared to have the same peptide backbone, since they showed a single band with the same mobility as a 160-kDa protein in SDS/PAGE and the same amino acid composition. However they differed from each other in having increasing iodine contents (1.9% to 5.9% by mass of total amino acids) associated with the increase in hormonal iodine proportion (8.4% to 16.7% of total iodine) and carbohydrate content (35.6% to 53.5% by mass). These values are strikingly different from those of thyroglobulin with an iodine content of less than 1%, hormonal iodine of 20-40% and carbohydrate content of less than 10%. The amino acid composition of the hagfish iodoprotein, especially the cysteine content of less than 1%, was also entirely different from that of thyroglobulin. These results suggest that most, if not all, tyrosine residues of the hagfish thyroid glycoprotein with a less rigid structure are susceptible to an iodinating system, but hormone residues are formed by a much less efficient mechanism than those in thyroglobulin, when poorly iodinated.     

Cloning, expression, and characterization of the unique bovine A1 adenosine receptor. Studies on the ligand binding site by site-directed mutagenesis.

The bovine brain A1 adenosine receptor (A1AR) is distinct from other A1ARs in that it displays the unique agonist potency series of N6-R-phenylisopropyladenosine (R-PIA) greater than N6-S-phenylisopropyladenosine (S-PIA) greater than 5'-N-ethylcarboxamidoadenosine and has a 5-10-fold higher affinity for both agonists and antagonists. The cDNA for this receptor has been cloned from a size-selected (2-4-kb) bovine brain library and sequenced. The 2.0-kb cDNA encodes a protein of 326 amino acid residues with a molecular mass of 36,570 daltons. The amino acid sequence fits well into the seven-transmembrane domain motif typical of G protein-coupled receptors. Northern analysis in bovine tissue using the full length cDNA demonstrates mRNAs of 3.4 and 5.7 kb with a tissue distribution consistent with A1AR binding. Subcloning of the cDNA in a pCMV5 expression vector with subsequent transfection into both COS7 and Chinese hamster ovary cells revealed a fully functional A1AR which could inhibit adenylylcyclase and retained the unique pharmacologic properties of the bovine brain A1AR. The A1AR was found to have a single histidine residue in each of transmembrane domains 6 and 7. Histidine residues have been postulated by biochemical studies to be important for ligand binding. Mutation of His-278 to Leu-278 (seventh transmembrane domain) dramatically decreased both agonist and antagonist binding by greater than 90%. In contrast, mutation of His-251 to Leu-251 decreased antagonist affinity and the number of receptors recognized by an antagonist radioligand. In contrast, agonist affinity was not perturbed but the number of receptors detected by an agonist radioligand was also reduced. These data suggest that both histidines are important for both agonist and antagonist binding, but His-278 appears critical for ligand binding to occur.     

Generation of deviation parameters for amino acid singlets, doublets and triplets from three-dimentional structures of proteins and its implications for secondary structure prediction from amino acid sequences

We present a new method, secondary structure prediction by deviation parameter (SSPDP) for predicting the secondary structure of proteins from amino acid sequence. Deviation parameters (DP) for amino acid singlets, doublets and triplets were computed with respect to secondary structural elements of proteins based on the dictionary of secondary structure prediction (DSSP)-generated secondary structure for 408 selected non-homologous proteins. To the amino acid triplets which are not found in the selected dataset, a DP value of zero is assigned with respect to the secondary structural elements of proteins. The total number of parameters generated is 15,432, in the possible parameters of 25,260. Deviation parameter is complete with respect to amino acid singlets, doublets, and partially complete with respect to amino acid triplets. These generated parameters were used to predict secondary structural elements from amino acid sequence. The secondary structure predicted by our method (SSPDP) was compared with that of single sequence (NNPREDICT) and multiple sequence (PHD) methods. The average value of the percentage of prediction accuracy for a helix by SSPDP, NNPREDICT and PHD methods was found to be 57%, 44% and 69% respectively for the proteins in the selected dataset. For b-strand the prediction accuracy is found to be 69%, 21% and 53% respectively by SSPDP, NNPREDICT and PHD methods. This clearly indicates that the secondary structure prediction by our method is as good as PHD method but much better than NNPREDICT method.     

Generation of deviation parameters for amino acid singlets, doublets and triplets from three-dimensional structures of proteins and its implications for secondary structure prediction from amino acid sequences

We present a new method, secondary structure prediction by deviation parameter (SSPDP) for predicting the secondary structure of proteins from amino acid sequence. Deviation parameters (DP) for amino acid singlets, doublets and triplets were computed with respect to secondary structural elements of proteins based on the dictionary of secondary structure prediction (DSSP)-generated secondary structure for 408 selected nonhomologous proteins. To the amino acid triplets which are not found in the selected dataset, a DP value of zero is assigned with respect to the secondary structural elements of proteins. The total number of parameters generated is 15,432, in the possible parameters of 25,260. Deviation parameter is complete with respect to amino acid singlets, doublets, and partially complete with respect to amino acid triplets. These generated parameters were used to predict secondary structural elements from amino acid sequence. The secondary structure predicted by our method (SSPDP) was compared with that of single sequence (NNPREDICT) and multiple sequence (PHD) methods. The average value of the percentage of prediction accuracy for αhelix by SSPDP, NNPREDICT and PHD methods was found to be 57%, 44% and 69% respectively for the proteins in the selected dataset. For Β-strand the prediction accuracy is found to be 69%, 21% and 53% respectively by SSPDP, NNPREDICT and PHD methods. This clearly indicates that the secondary structure prediction by our method is as good as PHD method but much better than NNPREDICT method.     

Differential effect of amino acid residues on the stability of double helices formed from polyribonucleotides and its possible relation to the evolution of the genetic code

The interaction of amino acid residues with polyribonucleotides was characterized by measurements of melting temperatures (tm) for poly(A).poly(U) and poly(I).poly(C) as functions of the concentrations of various amino acid amides. The amides of hydrophilic amino acids lead to a continuous increase of tm with increasing concentration, whereas amides of hydrophobic amino acids induce a decrease of tm at low concentrations (approximately 1 mM) followed by an increase at higher concentrations. Analysis of the data by a simple site model provides the affinity of each ligand for the double helix relative to that for the single strands. This parameter decreases in the order Ala greater than Gly greater than Ser greater than Asn greater than Pro greater than Met, Val greater than Ile, Leu for poly(A).poly(U) and Ala, Gly, Ser greater than Asn greater than Pro greater than Val greater than Ile, Met, Leu for poly(I).poly(C). The special effects of hydrophobic amino acids may be related to the similarity of the codons for these amino acids. A simple model for assignment of codons to amino acids is proposed.     

The structure of jack bean urease. The complete amino acid sequence, limited proteolysis and reactive cysteine residues

The amino acid sequence of jack bean urease has been determined. The protein consists of a single kind of polypeptide chain containing 840 amino acid residues. The subunit relative molecular mass calculated from the sequence is 90,770, indicating that urease is composed of six subunits. Out of 25 histidine residues in urease, 13 were crowded in the region between residues 479 and 607, suggesting that this region may contain the nickel-binding site. Limited tryptic digestion cleaved urease at two sites, Lys-128 and Lys-662. Proteolytic products were not dissociated and retained full enzymatic activity. Five tryptic peptides containing the reactive cysteine residues were isolated and characterized with the aid of sulfhydryl-specific reagents, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine and N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide. The reactive cysteine residues were located at positions 59, 207, 592, 663, and 824. The possibility that Cys-59, Cys-207, Cys-663, and Cys-824 are involved in the urease activity of the enzyme has been eliminated. Cys-592, which is essential for enzymatic activity, is located in the above-mentioned histidine-rich region.     

The primary structure of the cytotoxin restrictocin

The complete amino acid sequence of the single polypeptide chain of cytotoxin restrictocin has been determined. Its structure was established by automated Edman degradation of the intact molecule reduced and [14C]carboxymethylated and of fragments obtained by chemical cleavage of the protein with cyanogen bromide and BNPS-skatole and by enzymatic cleavage of the polypeptide chain with trypsin. The molecule consists of 149 amino acid residues with a calculated relative molecular mass of 16836. The protein presents two disulfide bridges, one between cysteine residues at positions 5 and 147 and the other one formed by cysteine residues at positions 75 and 131. The amino acid sequence of restrictocin shows a high degree of homology (86%) with that of the cytotoxin named alpha-sarcin.     

A superfamily of voltage-gated sodium channels in bacteria

NaChBac, a six-alpha-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na(+)-selective channel. As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na(+) channels (Na(V)PZ from Paracoccus zeaxanthinifaciens and Na(V)SP from Silicibacter pomeroyi). Na(V)PZ and Na(V)SP share approximately 40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both Na(V)PZ and Na(V)SP were Na(+)-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-alpha-helical transmembrane-spanning subunits constitute a widely distributed superfamily (Na(V)Bac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of Na(V)Bac structure and function of similarity and dissimilarity among Na(V)Bac proteins. Thus, the Na(V)Bac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.     

Gag proteins of the highly replicative MN strain of human immunodeficiency virus type 1: posttranslational modifications, proteolytic processings, and complete amino acid sequences.

The MN strain of human immunodeficiency virus type 1 was grown in H9 cells, concentrated by centrifugation, and disrupted, and proteins were purified by reversed-phase high-pressure liquid chromatography. Complete amino acid sequences were determined for the mature Gag proteins, showing natural proteolytic cleavage sites and the order of proteins (p17-p24-p2-p7-p1-p6) in the Gag precursors. At least two sequence variants of p24 and eight sequence variants of p17 were detected. The two most abundant variants of p24 and p17 represented at least 50% +/- 5% and 20% +/- 5% of their totals, respectively. These data suggest heterogeneity in the virus population, with 50% of the total virus containing the most abundant forms of p17 and p24 and 20% of the virus containing the second most abundant forms. The Gag precursors of these suggested viruses differ from each other by only 3 amino acid residues but differ from the precursors predicted by the published MN proviral DNA sequence by 10 residues. Electrospray ionization mass spectrometry analysis of the purified p24 forms showed that the measured molecular weight of the protein was 200 +/- 50 atomic mass units greater than the calculated molecular weight. The source of additional mass for the p24 forms was not determined, but the observation is consistent with previous suggestions that the protein is phosphorylated. Greater than 98% of the total recovered p17 was myristylated at the N-terminal glycine residue, and the measured molecular weights (as determined by electrospray ionization mass spectrometry) of the most abundant forms were within 3 atomic mass units of the calculated molecular weights (15,266).     

Acaloleptins A: inducible antibacterial peptides from larvae of the beetle, Acalolepta luxuriosa

We purified and characterized three structurally related antibacterial peptides with a molecular mass of 8 kDa (acaloleptins A1, A2, and A3) from the hemolymph of immunized larvae of the Udo longicorn beetle, Acalolepta luxuriosa. These peptides have the same 6 N-terminal amino acid residues and show potent antibacterial activity against some Gram-negative bacteria. The three peptides are thought to be isoforms. Reverse phase HPLC analysis of the hemolymph of immunized and naive larvae showed that acaloleptins A1, A2, and A3 were inducible and suggested that all three peptides were produced in a single insect. We determined the complete amino acid sequence of acaloleptin A1: Acaloleptin A1 consists of 71 amino acid residues and shares significant sequence similarity with coleoptericin and holotricin 2, which were isolated from other coleopteran insects. Furthermore, the 29 C-terminal residues of acaloleptin A1 had 40% identity with the 30 C-terminal residues of hymenoptaecin found in honeybees. Arch. Insect Biochem.     

Translocating proline-rich peptides from the antimicrobial peptide bactenecin 7

The intracellular delivery of most peptides, proteins, and nucleotides to the cytoplasm and nucleus is impeded by the cell membrane. To allow simplified, noninvasive delivery of attached cargo, cell-permeant peptides that are either highly cationic or hydrophobic have been utilized. Because cell-permeable peptides share half of the structural features of antimicrobial peptides containing clusters of charge and hydrophobic residues, we have explored antimicrobial peptides as templates for designing cell-permeant peptides. We prepared synthetic fragments of Bac 7, an antimicrobial peptide with four 14-residue repeats from the bactenecin family. The dual functions of cell permeability and antimicrobial activity of Bac 7 were colocalized at the N-terminal 24 residues of Bac 7. In general, long fragments of Bac(1-24) containing both regions were bactericidal and cell-permeable, whereas short fragments with only a cationic or hydrophobic region were cell-permeant without the attendant microbicidal activity when measured in a fluorescence quantitation assay and by confocal microscopy. In addition, the highly cationic fragments were capable of traversing the cell membrane and residing within the nucleus. A common characteristic shared by the cell-permeant Bac(1-24) fragments, irrespective of their number of charged cationic amino acids, is their high proline content. A 10-residue proline-rich peptide with two arginine residues was capable of delivering a noncovalently linked protein into cells. Thus, the proline-rich peptides represent a potentially new class of cell-permeant peptides for intracellular delivery of protein cargo. Furthermore, our results suggest that antimicrobial peptides may represent a rich source of templates for designing cell-permeant peptides.