Determination Of Amino Acid Sequence Research Articles

Rapid isolation and identification of staphylococcal exoproteins by reverse phase capillary high performance liquid chromatography-electrospray ionization mass spectrometry.

The isolation of staphylococcal extracellular toxins and enzymes (exoproteins) usually requires time-consuming purification steps such as repeated chromatographic separations and isoelectric focusing. We performed rapid isolation, quantification and identification of staphylococcal exoproteins by reverse phase capillary high performance liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) followed by the determination of N-terminal amino acid sequences of separated peaks. We identified two novel exoproteins as well as previously reported antigens ORF-1 and ORF-2, glutamyl endopeptidase in Staphylococcus aureus NCTC8325 and protein A, staphylococcal enterotoxin C3 (SEC3), toxic shock syndrome toxin-1 (TSST-1) and alpha-toxin in a clinical isolate methicillin-resistant S. aureus (MRSA) 3543. MRSA3543 secreted 5.33 and 1.45 microg of SEC3 and TSST-1 per 20 microg total exoproteins ml(-1), respectively. The capillary LC treatment of the exoprotein fraction separated at least 12 peaks, indicating its high-resolution power. We found that when a protein was once determined by its N-terminal sequence, its mass spectrum and the obtained molecular mass was applicable for the assignment of the protein.

Rapid isolation and identification of staphylococcal exoproteins by reverse phase capillary high performance liquid chromatography–electrospray ionization mass spectrometry

The isolation of staphylococcal extracellular toxins and enzymes (exoproteins) usually requires time-consuming purification steps such as repeated chromatographic separations and isoelectric focusing. We performed rapid isolation, quantification and identification of staphylococcal exoproteins by reverse phase capillary high performance liquid chromatography–electrospray ionization mass spectrometry (LC–ESI/MS) followed by the determination of N-terminal amino acid sequences of separated peaks. We identified two novel exoproteins as well as previously reported antigens ORF-1 and ORF-2, glutamyl endopeptidase in Staphylococcus aureus NCTC8325 and protein A, staphylococcal enterotoxin C3 (SEC3), toxic shock syndrome toxin-1 (TSST-1) and α-toxin in a clinical isolate methicillin-resistant S. aureus (MRSA) 3543. MRSA3543 secreted 5.33 and 1.45 μg of SEC3 and TSST-1 per 20 μg total exoproteins ml −1, respectively. The capillary LC treatment of the exoprotein fraction separated at least 12 peaks, indicating its high-resolution power. We found that when a protein was once determined by its N-terminal sequence, its mass spectrum and the obtained molecular mass was applicable for the assignment of the protein.

Purification and characterization of Sa-lrp, a DNA-binding protein from the extreme thermoacidophilic archaeon Sulfolobus acidocaldarius homologous to the bacterial global transcriptional regulator Lrp.

Archaea, constituting the third primary domain of life, harbor a basal transcription apparatus of the eukaryotic type, whereas curiously, a large fraction of the potential transcription regulation factors appear to be of the bacterial type. To date, little information is available on these predicted regulators and on the intriguing interplay that necessarily has to occur with the transcription machinery. Here, we focus on Sa-lrp of the extremely thermoacidophilic crenarchaeote Sulfolobus acidocaldarius, encoding an archaeal homologue of the Escherichia coli leucine-responsive regulatory protein Lrp, a global transcriptional regulator and genome organizer. Sa-lrp was shown to produce a monocistronic mRNA that was more abundant in the stationary-growth phase and produced in smaller amounts in complex medium, this down regulation being leucine independent. We report on Sa-Lrp protein purification from S. acidocaldarius and from recombinant E. coli, both identified by N-terminal amino acid sequence determination. Recombinant Sa-Lrp was shown to be homotetrameric and to bind to its own control region; this binding proved to be leucine independent and was stimulated at high temperatures. Interference binding experiments suggested an important role for minor groove recognition in the Sa-Lrp-DNA complex formation, and mutant analysis indicated the importance for DNA binding of the potential helix-turn-helix motif present at the N terminus of Sa-Lrp. The DNA-binding capacity of purified Sa-Lrp was found to be more resistant to irreversible heat inactivation in the presence of L-leucine, suggesting a potential physiological role of the amino acid as a cofactor.

Determination of the N-Terminal Amino Acid Sequence of the Purified Prothrombin from a Patient with Liver Cirrhosis

The reasons for the decreased functional activity of prothrombin in liver diseases are still speculative. When a highly purified preparation of prothrombin from a patient with liver cirrhosis is available, the cause of prothrombin abnormalities may be researched on a molecular basis. In this study, prothrombin (6.7 mg) was purified from the ascites fluid (1130 mL) of a patient with liver cirrhosis by barium citrate adsorption, ammonium sulfate elution, DEAE Sephacel and Heparin Sepharose CL-6B column chromatography steps. The molecular weight of this prothrombin was the same as that of normal prothrombin purified from a normal plasma pool. The specific activities were found to be 3.36 U/mg in the one stage clotting assay and 28.9 U/mg in the staphylocoagulase/chromogenic substrate assay, while the normal prothrombin specific activities were 3.92 U/mg and 30.1 U/mg respectively. When N-terminal amino acid sequence analysis was carried out, it was seen that the first 20 residues were identical to the normal human prothrombin excepting the Gla at position #14.

Did nucleotides or amino acids drive evolutionary conservation of the WT1 +/-KTS alternative splice?

Evolutionary comparisons frequently pinpoint crucial parts of a protein but, even within coding regions, nucleotides can do more than determine amino acid sequence. One highly conserved feature of the Wilms' tumour suppressor gene, WT1, is the potential, following alternative pre-mRNA splicing, to insert three amino acids (KTS) between the third and fourth zinc fingers. The nucleotides at this position simultaneously define amino acids and the alternative splice site. At the protein level this insertion influences DNA binding affinity and specificity, protein-protein interactions and subnuclear localization. Mutations within the +/-KTS splice junction lead to severe urogenital developmental abnormalities such as Frasier syndrome, indicating that the isoform ratio is critical for wild-type function. Using a series of site-directed mutations in both the genomic and cDNA context, the nucleotide-amino acid relationship was investigated. Mutational analysis within the cDNA suggests that the precise amino acids inserted may not be critical, but rather the disruption of the zinc finger structure alone may be sufficient to generate proteins with different in vitro properties. However, analysis within the genomic context suggests that the precise structure of the splice junction is crucial in retaining the balance between the isoforms, and this may account for the high nucleo-tide conservation of this unusual gene structure from fish to mammals.

An ichthyotoxic protein in the defensive skin secretion of the Red Sea trunkfish Ostracion cubicus

By the aid of acetone precipitation and RP-HPLC column chromatography coupled to various bioassays of ichthyotoxicity and cytolysis, a new ichthyotoxin designated “boxin” was isolated from the defensive skin secretion of the Red Sea trunkfish Ostracion cubicus. Boxin is a stable, heat and proteolysis resistant protein of 18 kDa. Its protein nature was assessed by spectral analysis, strong proteolysis, amino acid analysis and amino acid sequence determination. Similar to pahutoxin (PHN), an organic cationic surfactant derived from the same secretion, boxin is not effective by injection, and its ichthyotoxicity is achieved only upon delivery to the surrounding water. The latter suggests that fish lethality is mediated by externally allocated target sites (receptors). Boxin, however, differs from PHN by (1) possessing a 30 times higher ichthyotoxicity (mole per mole) and (2) being devoid of PHN's capacity to permeabilize biological membranes. From an ecological point of view, it is noteworthy that polypeptides are very useful in fulfilling allomonal functions in the marine environment due to the high information content inherent in their structures and their solubility in seawater.

A chloroplast envelope-transfer sequence functions as an export signal in Escherichia coli

The small subunit precursor of pea ribulose-1,5-bisphosphate carboxylase/oxygenase engineered with prokaryotic elements was expressed in Escherichia coli. This resulted in a dependable level of synthesis of the precursor protein in E. coli. The bacterially synthesised plant precursor protein was translocated from the cytoplasm and targeted to the outer membrane of the envelope zone. During the translocation step, a significant proportion of the precursor was processed to a soluble, mature SSU and found localised in the periplasm. The determined amino acid sequence of the isolated precursor showed that it had a deletion of an arginine residue at position −15 in the transit peptide. Expression of this transit peptide-appended mammalian cytochrome b 5 in E. coli displayed a targeting profile of the chromogenic chimera that was similar to that observed with the plant precursor protein.

Dipeptide Sequence Determination: Analyzing Phenylthiohydantoin Amino Acids by HPLC

Amino acid composition and sequence determination, important techniques for characterizing peptides and proteins, are essential for predicting conformation and studying sequence alignment. This experiment presents improved, fundamental methods of sequence analysis for an upper-division biochemistry laboratory. Working in pairs, students use the Edman reagent to prepare phenylthiohydantoin derivatives of amino acids for determination of the sequence of an unknown dipeptide. With a single HPLC technique, students identify both the N-terminal amino acid and the composition of the dipeptide. This method yields good precision of retention times and allows use of a broad range of amino acids as components of the dipeptide. Students learn fundamental principles and techniques of sequence analysis and HPLC.

Determination of amino acid sequence and site of mRNA expression of four vitellins in the giant freshwater prawn, Macrobrachium rosenbergii.

Four major yolk proteins, designated as vitellins (Vns) Macr-VnA, B, C, and D, were extracted from mature ovaries of Macrobrachium rosenbergii. These were purified to homogeneity by reversed-phase high performance liquid chromatography (HPLC) employing a unique separation system based on the hydrophobic properties of the Vn molecule. Using standard techniques of protein sequencing, more than 33 N-terminal and 57 internal amino acid residues were determined for each of the four Vns. The cDNA fragments encoding the four Vns were amplified by PCR using degenerate oligonucleotide primers derived from the N-terminal and internal amino acid sequences. These cDNA fragments were cloned, sequenced, and used as probes to examine the transcription of mRNAs encoding the four Vns. Significant accumulations of these mRNAs were observed in female hepatopancreas only, while mRNA expression was not detected in male hepatopancreas or any other female tissue including ovary, subepidermal adipose tissue, gill, and muscle. This is the first occasion in Crustacea in which multiple Vns were demonstrated to be synthesized simultaneously in a single tissue.

Pilot-scale purification of zeamatin, an antifungal protein from maize.

Zeamatin is an antifungal protein isolated from the seeds of Zea mays. A practical process for isolation and purification was developed to increase recovery yields for future testing of zeamatin as a novel therapeutic drug. Zeamatin was extracted with buffer from corn meal milled to flour. Solids were removed from the extract using pressure filtration, and zeamatin was purified using two separate reverse-phase chromatography steps. Determination of the amino-terminal amino acid sequence, Western blotting using anti-zeamatin antibody, and activity against Candida albicans were used to confirm that the purified protein was zeamatin. From 5 kg of corn meal, approximately 110 mg of zeamatin was purified to apparent homogeneity.

Biochemical characterization of 60S acidic ribosomal P proteins from porcine liver and the inhibition of their immunocomplex formation with sera from systemic lupus erythematosus (SLE) patients by glycyrrhizin in vitro.

The three casein kinase II (CK-II) phosphate acceptors (p35, p17 and p15) in the Superdex CK-II fraction prepared from a 1.5 M NaCl extract of porcine liver were selectively purified by glycyrrhizin (GL)-affinity column chromatography (HPLC) as a heterocomplex associated with CK-II. Determination of the N-terminal amino acid sequences and immunological tests confirmed that these three CK-II phosphate acceptors belong to the family of 60S acidic ribosomal proteins (P0, P1 and P2). Three polyphenol-containing anti-oxidant compounds [catechin, epigallocatechin gallate (EGCG) and quercetin] inhibited CK-II activity (phosphorylation of these ribosomal P proteins) in a dose-dependent manner in vitro. Quercetin (ID50 = approx. 50 nM) was found to be an effective CK-II inhibitor. In contrast, CK-II activity was significantly stimulated by lower doses (0.3-3 microl) of GL, but was inhibited at high doses above 30 microM. As expected, GL at high doses above 200 microM inhibited the immunocomplex formation of 60S acidic ribosomal P proteins with their specific antibodies in the sera from patients with systemic lupus erythematosus (SLE). These results suggest that (i) a GL-affinity column is useful for effective purification of 60S acidic ribosomal P proteins from various mammalian cells as a heterocomplex associated with CK-II; and (ii) a relative high dose of GL may prevent the immunocomplex formation of 60S acidic ribosomal P proteins with their specific antibodies in the sera of SLE patients.

Molecular cloning and characterization of a hemolymph clottable protein from tiger shrimp (Penaeus monodon).

To investigate the coagulation system in crustacean decapoda, a homodimeric glycoprotein of 380 kDa was purified from the hemolymph of tiger shrimp (Penaeus monodon) by sequential DEAE anion exchange chromatography. The purified protein was coagulated by the shrimp hemocyte transglutaminase in the presence of Ca2+. The clottable protein contains 44% alpha helices and 26% beta sheets as determined by circular dichroism spectra. Its conformation is stable in buffer of pH 4-9. To solve its primary structure, partial sequences of the purified polypeptides from cyanogen bromide cleavage and endopeptidase digestion were also determined. A shrimp cDNA expression library was constructed. By combination with antibody screening, reverse transcriptase PCR using degenerate primers from determined amino acid sequences and cDNA library screening with digoxigenin-labeled DNA probes, the entire cDNA of 6124 bp was obtained. This cDNA encodes a protein of 1670 amino acids, including a 14-amino acid signal peptide. With four potential N-glycosylation sites, the clottable protein was found to contain 3.8% high-mannose glycan; and Man8GlcNAc and Man9GlcNAc were released upon endo-beta-N-acetylglucosaminidase hydrolysis. Upon conducting a protein sequence database survey, the shrimp clottable protein shows 36% identities to the crayfish clotting protein and lower similarities to members of insect vitellogenins, apolipoprotein B and mammalian von Willebrand factor. Notably, a region rich in Gln residues, a polyGln motif and five Ser-Lys-Thr-Ser repeats are present in the shrimp protein, suggesting this protein might be a transglutaminase substrate. Northern blot analysis revealed that the clottable protein is expressed in most of the shrimp tissues but not in the mature hemocytes.

Identification of a Subtilisin-Like Protease in Seeds of Developing Tung Fruits

Summary A subtilisin-like protease was identified in the low-density microsomal fraction of developing tung seeds. The abundance of the protease changed significantly throughout seed development and showed a direct temporal correlation with accumulation of storage oil. Once storage oil synthesis was complete, the abundance of the protease decreased dramatically. Determination of the N-terminal amino acid sequence revealed that the protease sequence began just after a conserved pro-domain cleavage site, suggesting that the protease was fully active in developing tissue. Extraction of microsomal membranes with high salt or pH demonstrated that the protease was not a membrane-anchored protein.

Analysis of the kinase-related protein gene found at human chromosome 3q21 in a multi-gene cluster: Organization, expression, alternative splicing, and polymorphic marker

We report the amino acid sequence, genomic organization, tissue expression, and alternative splice patterns for the human kinase related protein (KRP) gene, as well as the discovery of a new CA repeat sequence polymorphic marker in an upstream intron of the myosin light chain kinase (MLCK) gene. The KRP/MLCK genetic locus is a prototype for a recently discovered paradigm in which an independently regulated gene for a non-enzymic protein is embedded within a larger gene for a signal transduction enzyme, and both classes of proteins are involved in the regulation of the same cellular structure. The MLCK/KRP gene cluster has been found only in higher vertebrates and is localized to human chromosome 3q21. The determination of the human KRP amino acid sequence through cDNA sequence analysis and its comparison to the exon/intron organization of the human KRP gene revealed an alternative splice pattern at the start of KRP exon 2, resulting in the insertion of a single glutamic acid in the middle of the protein. Examination of tissue distribution using Northern blot analysis revealed that the human expression pattern is more similar to the well-characterized chicken KRP gene expression pattern than to rodent or rabbit. Unexpected differences of the human gene from other species is the apparent expression of the human gene products in adult cardiac muscle, an observation that was pursued further by the production of a site-directed antiserum and immunohistochemistry analysis. The results reported here provide insight into the conserved and variable features of this late evolving genetic paradigm, raise new questions about the molecular aspects of cardiac muscle regulation, and provide tools needed for future clinical studies. The comparative analysis of the MLCK/KRP locus, combined with the recent discovery of a similar genomic relationship among other signal transduction proteins, suggest a diverse distribution of this theme among signal transduction systems in higher vertebrate genomes and indicate the utility of comparative genomics in revealing late evolving genetic paradigms.

Comparative Studies of High MrSubunits of Rye and Wheat. II. Partial Amino Acid Sequences

High M. subunit fractions of two rye cultivars (Danko and Halo) and of the wheat cultivar Rektor were isolated from defatted flours by extraction with 50% (v/v) aqucous propan-1-ol under reducing conditions and 60 °C followed by precipitation using a 60% concentration of propan-1-ol, centrifugation, dialysis and lyophilisation. Two single major subunits of rye Danko were isolated by to wheat high M r subunits. Total high M r subunit fractions of Danko, Halo and Rektor were digested with TPCK-treated trypsin at 37°C for 8h. The partial hydrolysates were pre-separated into the fractions I (M. 10 000) by centrifugation on a Centriprep 10-membranc. Both fractions were then separated by reversed-phase HPLC on C 18 , silica gel. Dominating peptides were purified by preparative rechromatography under slightly changed HPLC conditions and characterised by amino acid analysis and determination of amino acid sequences. The resulting sequences of 19 (Danko), 12 (Halo) and 15 peptides (Rektor) were compared with known sequences from x- and y-type high M r subunits of wheat. With the exception of three peptides, the sequences could be assigned to the three different domains of high M r subunits. Even when the sequences of the rye peptides were similar to those of wheat, they were not identical in any case; on average the variations were about 20%. Most of rye peptides corresponded to the x-type subunits of wheat. Within the most characteristic sequence motifs belonging to the repetitive domain B, fundamental differences between wheat and tye were not detected.

Dichain structure of botulinum neurotoxin: identification of cleavage sites in types C, D, and F neurotoxin molecules.

Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum as about a 150-kDa single-chain polypeptide. Posttranslational modification by bacterial or exogenous proteases yielded dichain structure which formed a disulfide loop connecting a 50-kDa light chain (Lc) and 100-kDa heavy chain (Hc). We determined amino acid sequences around cleavage sites in the loop region of botulinum NTs produced by type C strain Stockholm, type D strain CB16, and type F strain Oslo by analysis of the C-terminal sequence of Lc and the N-terminal sequence of Hc. Cleavage was found at one or two sites at Arg444/Ser445 and Lys449/Thr450 for type C, and Lys442/Asn443 and Arg445/Asp446 for type D, respectively. In culture fluid of mildly proteolytic strains of type C and D, therefore, NT exists as a mixture of at least three forms of nicked dichain molecules. The NT of type F proteolytic strain Oslo showed the Arg435 as a C-terminal residue of Lc and Ala440 as an N-terminal residue of Hc, indicating that the bacterial protease cuts twice (Arg435/Lys436 and Lys439/Ala440), with excision of four amino acid residues. The location of cleavage and number of amino acid residue excisions in the loop region could be explained by the degree of exposure of amino acid residues on the surface of the molecule, which was predicted as surface probability from the amino acid sequence. In addition, the observed correlation may also be adapted to the cleavage sites of the other botulinum toxin types, A, B, E, and G.

Determination of amino acid sequences of two subunits in sarcosine oxidase from Corynebacterium sp. U-96.

The primary structures of the C and D subunits of sarcosine oxidase from Corynebacterium sp. U-96 were determined by sequencing the peptide fragments derived from their enzymatic digestions. The C and D subunits were shown to be composed of 199 and 92 residues, respectively. Each amino acid sequence showed a high homology with the sequence of the corresponding subunit from Corynebacterium sp. P-1. However, there were some differences between these two species, that is, four N-terminal residues were truncated in the C subunit, but six C-terminal residues were truncated in the D subunit. The D subunit contained three cysteine residues, but no disulfide bonds are in the subunit. Overall sequences of both subunit showed no homology with any other protein in the data base.

Metal-dependent nucleotide binding to the Escherichia coli rotamase SlyD

Upon expression and purification of the first catalytic domain of mammalian adenylate cyclase type 1 (IC1), a 27 kDa contaminant was observed, which was labelled by three radioactive ATP analogues (8-azido-ATP, 3′-O-(4-benzoyl)benzoyl-ATP and 2′,3′-dialdehyde-ATP); the protein was purified separately and identified as Escherichiacoli SlyD by N-terminal amino acid sequence determination. SlyD is the host protein required for lysis of E.coli upon infection with bacteriophage ΦX174 and has recently been shown to display rotamase (peptidylproline cis-trans-isomerase) activity. The covalent incorporation of ATP analogues into SlyD was promoted by bivalent transition metal ions (Zn2+ ⩾ Ni2+ > Co2+ > Cu2+) but not by Mg2+ or Ca2+; this is consistent with the known metal ion specificity of SlyD. ATP, ADP, GTP and UTP suppressed labelling of SlyD with comparable potencies. Similarly, SlyD bound 2′,3′-O-(-2,4,6-trinitrophenyl)-ATP with an affinity in the range of 10 μM, as determined by fluorescence enhancement. This interaction was further augmented in the presence of Zn2+ (Kd = ∼ 2 μM at saturating Zn2+) but not of Mg2+. Irrespective of the assay conditions, hydrolysis of nucleotides by SlyD was not detected. Upon gel filtration on a Superose HR12 column, SlyD (predicted molecular mass = 21 kDa) migrated with an apparent molecular mass of 44 kDa, indicating that the protein was a dimer. However, the migration of SlyD was not affected by the presence of Zn2+ or of Zn2+ and ATP. Thus we concluded that SlyD binds nucleotides in the presence of metal ions. These findings suggest that SlyD serves a physiological role that goes beyond that accounted for by its intrinsic rotamase activity, which is observed in the absence of metal ions.

Amino acid sequence, crystallization and structure determination of reduced and oxidized cytochrome c6 from the green alga Scenedesmus obliquus

Cytochrome c 6 from the unicellular green alga Scenedesmus obliquus was sequenced, crystallized in its reduced and oxidized state and the three-dimensional structure of the protein in both redox states was determined by X-ray crystallography. Reduced cytochrome c 6 crystallized as a monomer in the space group P2 12 12, whereas the oxidized protein crystallized as a dimer in the space group P3 121. The structures were solved by molecular replacement and refined to 1.9 and 2.0 Å, respectively. Comparison of the structures of both redox states revealed only slight differences on the protein surface, whereas a distortion along the axis between the heme iron and its coordinating Met61 residue was observed. No redox-dependent movement of internal water molecules could be detected. The high degree of similarity of the surfaces and charge distributions of both redox states, as well as the dimerization of cytochrome c 6 as observed in the oxidized crystal, is discussed with respect to its biological relevance and its implications for the reaction mechanisms between cytochrome c 6 and its redox partners. The dimer of oxidized cytochrome c 6 may represent a molecular structure occurring in a binary complex with cytochrome b 6 f. This assembly might be required for the correct orientation of cytochrome c 6 with respect to its redox partner cytochrome b 6 f, facilitating the electron transfer within the complex. If the dimerization is not redox-dependent in vivo, the almost identical surfaces of both redox states do not support a long range differentiation between reduced and oxidized cyt c 6, i.e. a random collision model for the formation of an electron transfer complex must be assumed.

Duplicated Clostridium thermocellum cellobiohydrolase gene encoding cellulosomal subunits S3 and S5

The upstream region of the cellobiohydrolase gene cbhA of Clostridium thermocellum F7 was sequenced. It was found that this region contains the previously sequenced gene celK encoding an enzyme closely related to CbhA (cellulosomal subunit S3). The presence of a putative transcription terminator in the 524-bp intergenic region indicates that celK and cbhA are not cotranscribed as an operon. Sequence comparison between the two cellobiohydrolases revealed high sequence conservation in the catalytic domain and in the N-terminal cellulose-binding domain (CBD) homologous to CBD family IV, which binds specifically to amorphous cellulose and soluble cellooligosaccharides. In contrast to CbhA, CelK lacks a family III CBD capable of binding to crystalline cellulose. By partial amino acid sequence determination CelK was shown to be identical to cellulosomal subunit S5. CelK and CbhA were found to be members of subfamily E1 of cellulase family E (glycosylhydrolase family 9). Sequence comparison of catalytic domains of family E1 cellulases with C. thermocellum CelD, a family E1 endoglucanase of known three-dimensional structure, revealed a significant variation in the lengths of substrate-binding loops connecting the helices of the (alpha/alpha)6 barrel fold. The extended loops of CelK and CbhA might form an active-site tunnel, as found in the catalytic domains of fungal cellobiohydrolases.