The amino acid sequence of ferredoxin from Sambucus nigra

The amino acid sequence of the ferredoxin from Sambucus nigra consists of a single polypeptide chain of 97 amino acid residues, 5 of which are cysteine. The positions of the 4 cysteine residues which bind the iron atoms of the active centre are identical to those of other ferredoxins. Due to difficulties of obtaining pure protein, residues 87–90 have only been identified from the amino acid analysis of peptide C 10 and by homology with other higher plant ferredoxins.     

Amino acid pair- and triplet-wise groupings in the interior of α-helical segments in proteins

A statistical approach has been applied to analyse primary structure patterns at inner positions of α-helices in proteins. A systematic survey was carried out in a recent sample of non-redundant proteins selected from the Protein Data Bank, which were used to analyse α-helix structures for amino acid pairing patterns. Only residues more than three positions apart from both termini of the α-helix were considered as inner. Amino acid pairings i, i+k (k=1, 2, 3, 4, 5), were analysed and the corresponding 20×20 matrices of relative global propensities were constructed. An analysis of (i, i+4, i+8) and (i, i+3, i+4) triplet patterns was also performed. These analysis yielded information on a series of amino acid patterns (pairings and triplets) showing either high or low preference for α-helical motifs and suggested a novel approach to protein alphabet reduction. In addition, it has been shown that the individual amino acid propensities are not enough to define the statistical distribution of these patterns. Global pair propensities also depend on the type of pattern, its composition and orientation in the protein sequence. The data presented should prove useful to obtain and refine useful predictive rules which can further the development and fine-tuning of protein structure prediction algorithms and tools.     

Intramolecular interactions in pancreatic ribonucleases.

A detailed analysis of the composition and properties of hydrophobic nuclei and microclusters in pancreatic ribonuclease A (RNase A) has been carried out. Distance calculations for all noncovalently bonded atoms revealed that the average number of nonpolar contacts between a side chain of an amino acid and its neighbors is substantially larger if it involves hydrophobic residues rather than nonhydrophobic ones. However, the difference decreased when the number of contacts per nonpolar group and/or atom were calculated. Three main nuclei and five microclusters were identified, and their quantitative parameters were calculated. These nuclei include hydrophobic residues with a substantial number of nonpolar contacts with the environment (Phe 8, Phe 120, Phe 46, Tyr 25, Tyr 97, Ile 107, Leu 35, Ile 81, Val 54, Val 108, Met 29, Met 30). Hydrophobic nuclei of RNase A differ in shape and in composition, in the number of intranuclear contacts and of associated residues, as well as in their internal mobility. All eight cysteine residues are involved in nonpolar interactions with amino acid residues of hydrophobic nuclei. Active site amino acid residues of RNase A form a noncovalent contact network comprised of themselves, as well as of many conserved residues from hydrophobic nuclei. Sequence alignment with some other members of the RNase A family of proteins shows remarkable similarity in positions and in conservation of the main nonpolar residues, comprising cores of two (out of three) hydrophobic nuclei. A correlation was shown to exist between the average density of contacts for side-chain atoms and the number of amino acids to be found in the appropriate positions in the sequences of related mammalian ribonucleases. However, there are certain amino acid positions in the third, smaller nucleus, which are highly variable within the family. Taking into account that this nucleus is composed of residues belonging to different elements of the secondary structure, it is likely that the mutual orientation of these elements can be somehow different for these proteins.     

The amino acid composition of proteins: a method of analysis.

Four quasiloglinear models are proposed for describing relationships between the amino acid composition of proteins and the structure of the genetic code. The models allow estimation of base frequencies in all three codon positions and can be used to investigate “interactions” between any two codon positions. The estimation procedure proposed by Ohta and Kimura (Genetics64 (1970), 387–395) is discussed and using two of the proposed quasiloglinear models an analysis of the amino acid composition of human cytochrome c is presented. The analysis suggests that of the six codons which code for leucine (CUU, CUC, CUA and CUG) do not occur in human cytochrome c.     

The amino acid sequence of Phaseolus aureus L. (mung-bean) cytochrome c

The amino acid sequence of Phaseolus aureus L. (mung-bean) cytochrome c has been determined. The molecule consists of a single polypeptide chain of 111 amino acid residues and is homologous with other mitochondrial cytochromes c. Comparison with the amino acid sequence of wheat-germ cytochrome c (Stevens, Glazer & Smith, 1967) shows 14 differences. On alignment with mammalian cytochromes c, mung-bean cytochrome c has an N-acetylated ;tail' of eight amino acid residues similar to that found in wheat-germ cytochrome c. Of the 22 positions in wheat-germ cytochrome c that contain amino acid residues unique to these positions, 20 were found to contain the same ones in mung-bean cytochrome c. The in-N-trimethyl-lysine residues reported for wheat-germ cytochrome c (Delange, Glazer & Smith, 1969) in positions 72 and 86 were also found in these positions in mung-bean cytochrome c. The sequence was determined from 3mumol, by using chymotryptic and tryptic peptides which were analysed by the ;dansyl'-Edman method (Gray & Hartley, 1963a), with confirmation by amino acid analysis.     

The complete amino acids sequence of human complex-forming glycoprotein heterogeneous in charge (protein HC)

The complete amino acid sequence of human protein HC isolated from the urine of a single individual (JL) was determined. The polypeptide chain contained 181 residues, had a calculated molecular weight of 20,435 and contained 3 cysteine residues at positions 34, 70 and 167. An intrachain disulfide bridge was formed by residues 70 and 167. No variation of the amino acid sequence of protein HC was found and can therefore not explain the charge heterogeneity of protein HC in a given individual. The amino acid sequence of protein HC was almost identical to the one reported for human α₁-microglobulin but contained 14 additional residues.     

Complex Patterns of Histidine, Hydroxylated Amino Acids and the GxxxG Motif Mediate High-affinity Transmembrane Domain Interactions

Specific interactions of transmembrane helices play a pivotal role in the folding and oligomerization of integral membrane proteins. The helix-helix interfaces frequently depend on specific amino acid patterns. In this study, a heptad repeat pattern was randomized with all naturally occurring amino acids to uncover novel sequence motifs promoting transmembrane domain interactions. Self-interacting transmembrane domains were selected from the resulting combinatorial library by means of the ToxR/POSSYCCAT system. A comparison of the amino acid composition of high-and low-affinity sequences revealed that high-affinity transmembrane domains exhibit position-specific enrichment of histidine. Further, sequences containing His preferentially display Gly, Ser, and/or Thr residues at flanking positions and frequently contain a C-terminal GxxxG motif. Mutational analysis of selected sequences confirmed the importance of these residues in homotypic interaction. Probing heterotypic interaction indicated that His interacts in trans with hydroxylated residues. Reconstruction of minimal interaction motifs within the context of an oligo-Leu sequence confirmed that His is part of a hydrogen bonded cluster that is brought into register by the GxxxG motif. Notably, a similar motif contributes to self-interaction of the BNIP3 transmembrane domain.     

Structural position correlation analysis (SPCA) for protein family

Background

The proteins in a family, which perform the similar biological functions, may have very different amino acid composition, but they must share the similar 3D structures, and keep a stable central region. In the conservative structure region similar biological functions are performed by two or three catalytic residues with the collaboration of several functional residues at key positions. Communication signals are conducted in a position network, adjusting the biological functions in the protein family.

Methodology

A computational approach, namely structural position correlation analysis (SPCA), is developed to analyze the correlation relationship between structural segments (or positions). The basic hypothesis of SPCA is that in a protein family the structural conservation is more important than the sequence conservation, and the local structural changes may contain information of biology functional evolution. A standard protein P⁽⁰⁾ is defined in a protein family, which consists of the most-frequent amino acids and takes the average structure of the protein family. The foundational variables of SPCA is the structural position displacements between the standard protein P⁽⁰⁾ and individual proteins P_i of the family. The structural positions are organized as segments, which are the stable units in structural displacements of the protein family. The biological function differences of protein members are determined by the position structural displacements of individual protein P_i to the standard protein P⁽⁰⁾. Correlation analysis is used to analyze the communication network among segments.

Conclusions

The structural position correlation analysis (SPCA) is able to find the correlation relationship among the structural segments (or positions) in a protein family, which cannot be detected by the amino acid sequence and frequency-based methods. The functional communication network among the structural segments (or positions) in protein family, revealed by SPCA approach, well illustrate the distantly allosteric interactions, and contains valuable information for protein engineering study.     

A novel 7-β-(4-carboxybutanamido)-cephalosporanic acid acylase isolated from Pseudomonas strain C427 and its high-level production in Escherichia coli

We cloned the gene for 7-β-(4-carboxybutanamido)-cephalosporanic acid (GL-7ACA) acylase from Pseudomonas strain C427. The DNA sequence revealed an open reading frame of 2154 bp coding for 718 amino acid residues. The deduced amino acid sequence consists of 4 structural domains: (i) a signal peptide (positions 1–27), (ii) a small subunit of the acylase (positions 28–190), designated as α, (iii) a spacer peptide (positions 191–198), (iv) a large subunit (positions 199–718), designated as β. Plasmids were constructed to direct the synthesis of the acylase in Escherichia coli and the following results were obtained. The active acylase consists of two subunits which are processed from a single precursor protein, removing the spacer peptide during processing. A proportion of active acylase is secreted into the periplasm and the remainder is retained in the cytoplasm. The amount of precursor protein accumulated in the cytoplasm is greatly reduced when plasmids for the acylase lacking the signal sequence are expressed. Therefore, processing is independent of the translocation of the gene product through the cytoplasmic membrane, in contrast to the situation for penicillin G acylase. A high level of active enzyme production was achieved with a plasmid coding for an acylase in which the amino terminal sequence (positions 1–32) of native acylase is replaced by MFPTT.     

The amino acid sequences of plastocyanin from Mercurialis perennis and Capsella bursa-pastoris

The amino acid sequences of the plastocyanins from Mercurialis perennis and Capsella bursa-pastoris have been determined. The amides at positions 64 and 68 in the Mercurialis sequence were positioned by ‘homology’ Both proteins are single polypeptide chains of 99 residues and are closely related to other higher plant plastocyanins.     

Relationship between structure and amino acid sequence of strongly twisted and coiled β-hairpins in globular proteins

β-Hairpins are widespread in proteins, and it is possible to find them both within β-sheets and separately. In this work, a comparative analysis of amino acid sequences of β-strands within strongly twisted β-hairpins from different structural protein subclasses has been conducted. Strongly twisted and coiled β-hairpin generates in the space a right double helix out of β-strands that are connected by a loop region (connections). The frequencies of amino acid residues on the internal (concave) and external (convex) surfaces of strongly twisted β-hairpins have been determined (220 β-hairpins from nonhomologous proteins were studied). The concave surface of these β-hairpins is mainly generated by hydrophobic residues, while the convex surface by hydrophilic residues; accordingly, the alternation of hydrophobic internal and hydrophilic external residues is observed in their amino acid sequences. Amino acid residues of glycine and alanine (especially in places of the largest twisting of the strands) were anomalously frequently found in internal positions of strongly twisted and coiled β-hairpins. It was established that internal positions never contain the proline residues, while external positions in the twisting region contain them in a relatively large amount. It was demonstrated that at least one amino acid residue in α_L- or ε-conformation is required for generation of relatively short (up to 7 amino acid residues) connection. As a rule, these positions are occupied by glycines. Thus, not only the alternation of hydrophobic and hydrophilic amino acid residues, but also the presence of one or two glycine residues in the connection region and the excess of glycines and alanines in the places of the largest strand twisting on the concave surface, as well as the presence of prolines on the convex surface, are required to generate a strongly twisted and coiled β-hairpin.     

Arrangement of disulfide bridges and positions of sulfhydryl groups in tetanus toxin

Tetanus toxin is a 151-kDa protein. The complete amino acid sequence is known. The mature toxin is made up of two peptide chains and contains 10 half-cystine residues. Treatment with 4-vinylpyridine in the presence of 6 M guanidine converted six of them into S-pyridylethyl cysteine residues as determined by amino acid analysis. When alkylation was preceded by mercaptolysis, all 10 half-cystine residues were recovered in the S-pyridylethylated form. It was therefore concluded that the toxin contains six sulfhydryl groups and two disulfide bonds. The positions of the residues carrying sulfhydryl groups and of those involved in disulfide bridges were determined by labelling of the toxin alternatively with 4-vinylpyridine or with 4-dimethylaminoazobenzene-4'-iodoacetamide (DABIA), directly or after mercaptolysis. The toxin derivatives were cleaved with cyanogen bromide and the elution patterns in reversed-phase HPLC compared. The chromatography components were identified by N-terminal amino acid sequence and amino acid composition. In the chromatography of the non-mercaptolysed, DABIA-treated sample four chromophore-carrying components were detected which could be demonstrated by N-terminal sequence analysis to correspond to six half-cystine-containing cyanogen bromide fragments. In the mercaptolysed, DABIA-treated sample three additional chromophore-carrying components were present, corresponding to two previously disulfide-linked cyanogen bromide fragments and one fragment which had contained an internal disulfide bridge. The HPLC patterns showed characteristic differences as the DABIA-labelled fragments were considerably more hydrophobic than the corresponding vinylpyridine-labelled fragments. It was established that the half-cystine residues in positions 26, 185, 198, 311, 868, and 1300 are present in the sulfhydryl form, that those in positions 438 and 466 are disulfide-bridged, thereby connecting the light and heavy chains of the toxin, and that those in positions 1076 and 1092 are disulfide-bridged, thereby giving rise to a loop in the heavy chain. During the progress of the investigations about 20% of the amino acid sequence previously predicted from DNA analysis was confirmed by protein-chemical methods.     

The mucilage of Opuntia ficus-indica

The mucilage extracted from the cladodes (modified stems) of Opuntia ficus-indica contains residues of d-galactose, d-xylose, l-arabinose, l-rhamnose, and d-galacturonic acid. Seasonal variation in the sugar composition of the mucilage has been investigated. Fractionation studies indicate that the mucilage is essentially homogeneous. The rate of release of the constituent sugars and the change in optical rotation on mild hydrolysis coupled with the results of chromic acid oxidation suggest that the mucilage contains α-arabinofuranosyl, β-xylopyranosyl, β-rhamnopyranosyl, β-galactopyranosyl, and α-galactopyranosyluronic acid residues. The results also suggest a core containing galacturonic acid, rhamnose, and galactose, to which xylose and arabinose are attached in peripheral positions.     

Evidence for the amino-acid sequence of Crithidia fasciculata Cytochrome c555.

Evidence is presented which indicates that the amino acid sequence of cytochrome c555 from Crithidia fasciculata differs at sixteen positions from that of cytochrome c557 from Crithidia oncopelti. 101 residues were identified by dansyl-Edman degradation, carboxypeptidase digestion or considerations of the specificity of trypsin and of these, thirteen were found to be different from C. oncopelti cytochrome c557. The remaining 11 residues found in the amino acid composition of the trypic peptides were aligned on the basis of homology with cytochrome c557 and three further differences are proposed. The total of sixteen amino acid differences is surprising in view of the morphological and biochemical similarities of these organisms, and illustrates the problem of taxonomy of morphologically simple organisms. In both cytochromes only one cysteine residue is involved in the attachment of the protein to the prosthetic group.     

Comparative chemical studies of the polyhedral proteins of the nuclear polyhedrosis viruses of Bombyx mori and Galleria mellonella

Amino and carboxyl terminal groups, amino acid composition, and peptide maps of polyhedral proteins of the nuclear polyhedrosis viruses (NPV) of Bombyx mori and Galleria mellonella were investigated. It is shown that both the proteins have a tyrosine residue as their carboxyl terminal group and no amino terminal group. Amino acid compositions of the proteins are similar. The proteins are found to have 242 residues. From the amino acid composition, a molecular weight of 28,000 was calculated. The tryptic peptide maps of both the proteins differed only in a few peptides.It is inferred that the polyhedral proteins of B. mori and G. mellonella NPV have a closely similar primary structure.     

Characterizing the N-terminal processing motif of MHC class I ligands

Most peptide ligands presented by MHC class I molecules are the product of an intracellular pathway comprising protein breakdown in the cytosol, transport into the endoplasmic reticulum, and successive N-terminal trimming events. The efficiency of each of these processes depends on the amino acid sequence of the presented ligand and its precursors. Thus, relating the amino acid composition N-terminal of presented ligands to the sequence specificity of processes in the pathway gives insight into the usage of ligand precursors in vivo. Examining the amino acid composition upstream the true N terminus of MHC class I ligands, we demonstrate the existence of a distinct N-terminal processing motif comprising approximately seven residues and matching the known preferences of proteasome and TAP, two key players in ligand processing. Furthermore, we find that some residues, which are preferred by both TAP and the proteasome, are underrepresented at positions immediately preceding the N terminus of MHC class I ligands. Based on experimentally determined aminopeptidase activities, this pattern suggests trimming next to the final N terminus to take place predominantly in the endoplasmic reticulum.     

Plasmodium chitinases: revisiting a target of transmission‐blockade against malaria

Malaria is a life‐threatening disease caused by one of the five species of Plasmodium, among which Plasmodium falciparum cause the deadliest form of the disease. Plasmodium species are dependent on a vertebrate host and a blood‐sucking insect vector to complete their life cycle. Plasmodium chitinases belonging to the GH18 family are secreted inside the mosquito midgut, during the ookinete stage of the parasite. Chitinases mediate the penetration of parasite through the peritrophic membrane, facilitating access to the gut epithelial layer. In this review, we describe Plasmodium chitinases with special emphasis on chitinases from P. falciparum and P. vivax, the representative examples of the short and long forms of this protein. In addition to the chitinase domain, chitinases belonging to the long form contain a pro‐domain and chitin‐binding domain. Amino acid sequence alignment of long and short form chitinase domains reveals multiple positions containing variant residues. A subset of these positions was found to be conserved or invariant within long or short forms, indicating the role of these positions in attributing form‐specific activity. The reported differences in affinities to allosamidin for P. vivax and P. falciparum were predicted to be due to different residues at two amino acid positions, resulting in altered interactions with the inhibitor. Understanding the role of these amino acids in Plasmodium chitinases will help us elucidate the mechanism of catalysis and the mode of inhibition, which will be the key for identification of potent inhibitors or antibodies demonstrating transmission‐blocking activity.     

Evidence for Production of a New Lantibiotic (Butyrivibriocin OR79A) by the Ruminal Anaerobe Butyrivibrio fibrisolvens OR79: Characterization of the Structural Gene Encoding Butyrivibriocin OR79A

The ruminal anaerobe Butyrivibrio fibrisolvens OR79 produces a bacteriocin-like activity demonstrating a very broad spectrum of activity. An inhibitor was isolated from spent culture fluid by a combination of ammonium sulfate and acidic precipitations, reverse-phase chromatography, and high-resolution gel filtration. N-terminal analysis of the isolated inhibitor yielded a 15-amino-acid sequence (G-N/Q-G/P-V-I-L-X-I-X-H-E-X-S-M-N). Two different amino acid residues were detected in the second and third positions from the N terminus, indicating the presence of two distinct peptides. A gene with significant homology to one combination of the determined N-terminal sequence was cloned, and expression of the gene was confirmed by Northern blotting. The gene (bvi79A) encoded a prepeptide of 47 amino acids and a mature peptide, butyrivibriocin OR79A, of 25 amino acids. Significant sequence homology was found between this peptide and previously reported lantibiotics containing the double-glycine leader peptidase processing site. Immediately downstream of bvi79A was a second, partial open reading frame encoding a peptide with significant homology to proteins which are believed to be involved in the synthesis of lanthionine residues. These findings indicate that the isolated inhibitory peptides represent new lantibiotics. Results from both total and N-terminal amino acid sequencing indicated that the second peptide was identical to butyrivibriocin OR79A except for amino acid substitutions in positions 2 and 3 of the mature lantibiotic. Only a single coding region was detected when restriction enzyme digests of total DNA were probed either with an oligonucleotide based on the 5′ region of bvi79A or with degenerate oligonucleotides based on the predicted sequence of the second peptide.     

The amino acid sequence of ferredoxin from the alga Mastigocladuslaminosus

Ferredoxin was purified from the thermophilic blue-green alga, Mastigocladuslaminosus. The physicochemical properties of this ferredoxin are similar to those of other [2Fe-2S] plant ferredoxins except for its unusual thermal stability. The primary structure of the protein was determined and consists of 98 amino acid residues, 5 of which are cysteines. The positions of 4 cysteines which bind the iron atoms of the active centre are identical to those in other ferredoxins. The primary structure of the ferredoxin does not reveal any special features to account for its high thermal stability.