Amino acid sequence of rabbit skeletal muscle alpha-tropomyosin. The COOH-terminal half (residues 142 to 284)

Amino acid sequence analysis of the large cyanogen bromide fragment (residues 142 to 281) derived from the COOH-terminal half of the mixed tropomyosin population of rabbit skeletal muscle has been carried out. The isolation and sequence analysis of peptides derived from chymotryptic digests and from tryptic digests of the maleylated fragment permitted the alignment of the complete sequence except for the assignment of acids or amides at residues 142, 144, and 145. Selected peptides from a Myxobacter 495 alpha-lytic protease digest have confirmed certain overlaps. Based on previously published data the sequence can be extended to residue 284, the COOH-terminal end of the protein. In fourteen positions, amino acid substitutions have been observed. In one of these (residue 199) the sequence evidence indicates a minimum of four different polypeptide chains in the mixed tropomyosin population. The assignment of particular amino acid residues to these positions for the major alpha-component of rabbit skeletal tropomyosin has been based on the relative recoveries of peptides containing different residues in these positions.     

Arabidopsis cDNA encoding a membrane-associated protein with an acyl-CoA binding domain

Acyl-CoA binding proteins (ACBPs) are small (ca. 10 kDa) highly-conserved cytosolic proteins that bind long-chain acyl-CoAs. A novel cDNA encoding ACBP1, a predicted membrane protein of 24.1 kDa with an acyl-CoA binding protein domain at its carboxy terminus, was cloned from Arabidopsis thaliana. At this domain, ACBP1 showed 47% amino acid identity to Brassica ACBP and 35% to 40% amino acid identity to yeast, Drosophila, bovine and human ACBPs. Recombinant (His)6-ACBP1 fusion protein was expressed in Escherichia coli and was shown to bind 14[C]oleoyl-CoA. A hydrophobic domain, absent in the 10 kDa ACBPs, was located at the amino terminus of ACBP1. Using antipeptide polyclonal antibodies in western blot analysis, ACBP1 was shown to be a membrane-associated glycosylated protein with an apparent molecular mass of 33 kDa. The ACBP1 protein was also shown to accumulate predominantly in siliques and was localized to the seed within the silique. These results suggest that the biological role of ACBP1 is related to lipid metabolism in the seed, presumably in which acyl-CoA esters are involved. Northern blot analysis showed that the 1.4 kb ACBP1 mRNA was expressed in silique, root, stem, leaf and flower. Results from Southern blot analysis of genomic DNA suggest the presence of at least two genes encoding ACBPs in Arabidopsis.     

Amino acid sequence of a phosphorylation site in skeletal muscle glycogen synthetase.

Rabbit skeletal muscle glycogen synthetase was phosphorylated by incubation with [γ-³²P]ATP, Mg⁺⁺ and cyclic AMP-dependent protein kinase catalytic subunit from the same source. One of the major phosphorylation site peptides was isolated following brief tryptic-hydrolysis, and shown to have the sequence

     

Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells.

The whole amino acid sequence of nidogen was deduced from cDNA clones isolated from expression libraries and confirmed to approximately 50% by Edman degradation of peptides. The protein consists of some 1217 amino acid residues and a 28-residue signal peptide. The data support a previously proposed dumb-bell model of nidogen by demonstrating a large N-terminal globular domain (641 residues), five EGF-like repeats constituting the rod-like domain (248 residues) and a smaller C-terminal globule (328 residues). Two more EGF-like repeats interrupt the N-terminal and terminate the C-terminal sequences. Weak sequence homologies (25%) were detected between some regions of nidogen, the LDL receptor, thyroglobulin and the EGF precursor. Nidogen contains two consensus sequences for tyrosine sulfation and for asparagine beta-hydroxylation, two N-linked carbohydrate acceptor sites and, within one of the EGF-like repeats an Arg-Gly-Asp sequence. The latter was shown to be functional in cell attachment to nidogen. Binding sites for laminin and collagen IV are present on the C-terminal globule but not yet precisely localized.     

Amino acid sequence of an active fragment of rabbit skeletal muscle myosin light chain kinase

The amino acid sequence of a 368-residue segment at the carboxyl-terminus of rabbit skeletal muscle myosin light chain kinase (MLCK) has been determined. The sequence was derived primarily from analysis of two complementary sets of fragments obtained by cleavage at methionyl and arginyl bonds in S-carboxymethylated MLCK. The segment included a 360-residue fragment produced by limited tryptic digestion of MLCK. This fragment was both catalytically active and dependent on Ca2+-calmodulin. Unique structural features of MLCK have been identified, and a likely calmodulin interaction site is suggested. Sequence comparisons of MLCK to other protein kinases indicate close structural relationships in spite of marked differences in physicochemical properties, enzymatic characteristics, and regulatory response among these enzymes.     

The major myosin-binding domain of skeletal muscle MyBP-C (C protein) resides in the COOH-terminal, immunoglobulin C2 motif

A common feature shared by myosin-binding proteins from a wide variety of species is the presence of a variable number of related internal motifs homologous to either the Ig C2 or the fibronectin (Fn) type III repeats. Despite interest in the potential function of these motifs, no group has clearly demonstrated a function for these sequences in muscle, either intra- or extracellularly. We have completed the nucleotide sequence of the fast type isoform of MyBP-C (C protein) from chicken skeletal muscle. The deduced amino acid sequence reveals seven Ig C2 sets and three Fn type III motifs in MyBP-C. alpha-chymotryptic digestion of purified MyBP-C gives rise to four peptides. NH2-terminal sequencing of these peptides allowed us to map the position of each along the primary structure of the protein. The 28-kD peptide contains the NH2-terminal sequence of MyBP-C, including the first C2 repeat. It is followed by two internal peptides, one of 5 kD containing exclusively spacer sequences between the first and second C2 motifs, and a 95-kD fragment containing five C2 domains and three fibronectin type III motifs. The C-terminal sequence of MyBP-C is present in a 14- kD peptide which contains only the last C2 repeat. We examined the binding properties of these fragments to reconstituted (synthetic) myosin filaments. Only the COOH-terminal 14-kD peptide is capable of binding myosin with high affinity. The NH2-terminal 28-kD fragment has no myosin-binding, while the long internal 100-kD peptide shows very weak binding to myosin. We have expressed and purified the 14-kD peptide in Escherichia coli. The recombinant protein exhibits saturable binding to myosin with an affinity comparable to that of the 14-kD fragment obtained by proteolytic digestion (1/2 max binding at approximately 0.5 microM). These results indicate that the binding to myosin filaments is mainly restricted to the last 102 amino acids of MyBP-C. The remainder of the molecule (1,032 amino acids) could interact with titin, MyBP-H (H protein) or thin filament components. A comparison of the highly conserved Ig C2 domains present at the COOH- terminus of five MyBPs thus far sequenced (human slow and fast MyBP-C, human and chicken MyBP-H, and chicken MyBP-C) was used to identify residues unique to these myosin-binding Ig C2 repeats.     

Amino acid sequence of alpha chain of sarcoplasmic calcium binding protein obtained from shrimp tail muscle

The isotypes of sarcoplasmic Ca2+ binding protein (SCP) were purified from shrimp tail muscle. SCP exists in a dimeric form. One sample of shrimp contained only alpha A chain, whereas another contained alpha B and beta chains, and a heterodimer of alpha B beta which was not analyzed precisely. The amino acid sequences of the two alpha chains were determined. The two alpha chains are composed of 190 and 192 amino acid residues, respectively. The sequences of the two alpha chains differed in only four amino acids out of 192 residues. The sequences indicate that the alpha chain has three Ca2+-binding sites which are common to EF-hand type Ca2+-binding protein. In the absence of added Ca2+ and Mg2+, the amounts of bound Ca2+ in alpha A, alpha B, and beta chains were 3.0, 3.3, and 2.4 mol/22,000 g protein, respectively. Thus, it is suggested that all three isotypes of shrimp SCP have three Ca2+-binding sites which have high affinity to Ca2+. The sequence homology of shrimp SCP with other EF-hand type Ca2+-binding proteins is very low. The protein having the greatest homology with this SCP was cod parvalbumin; the sequence homology is 18%.     

Gene overlap results in a viral protein having an RNA binding domain and a major coat protein domain

L-A double-stranded RNA (dsRNA) replicates in vivo in yeast in a conservative, asynchronous (first [+] strand then [-] strand), intraviral process. New particles are formed by packaging (+) strands. Added viral (+) single-stranded RNA (ssRNA) is specifically bound by empty virus-like particles (VLPs) and, in a reaction requiring a host factor, is converted in vitro to dsRNA. We find that the isolated binding complex replicates only if it was formed in the presence of the host factor. The VLP minor 180 kd protein, but not the major coat protein, has ssRNA binding activity on Western blots. The 180 kd protein shares a common antigenic domain with the major coat protein, the latter known to be encoded by L-A dsRNA. The 180 kd protein, but not the major coat protein, also shares an antigenic domain with a sequence encoded by the 3' end of the L-A (+) strand. Thus the 180 kd protein is also encoded by L-A dsRNA and consists of a major coat protein domain and a ssRNA binding domain.     

Amino acid sequence of UP1, an hnRNP-derived single-stranded nucleic acid binding protein from calf thymus

The UP1 single-stranded nucleic acid binding protein from calf thymus (Herrick, G. & Alberts, B.M. (1976) J. Biol. Chem. 251, 2124-2132) has recently been shown to be a proteolytic fragment derived from the A1 heterogeneous nuclear ribonucleoprotein (hnRNP) (Pandolfo et al. (1985) Nucleic Acids Res. 13, 6577-6590). The NH2-terminus of the 22,162 dalton UP1 protein appears to be blocked, which suggests that UP1 represents the NH2-terminal two thirds of this 32,000 dalton hnRNP protein. The complete amino acid sequence for UP1 was derived from automated sequencing of peptides that were purified by HPLC from digests with trypsin, chymotrypsin, Staphylococcus aureus protease, endoproteinase Lys-C, and cyanogen bromide. Trichloroacetic acid precipitation followed by enzymatic digestion in 2 M urea proved to be the best approach for generating UP1 peptides. By carboxymethylating after, rather than before, digestion it was possible to avoid problems associated with the insolubility of the carboxymethylated UP1. All of the resulting peptides in amounts varying from 2 to 15 nmol were coupled to aminopolystyrene prior to solid-phase sequencing. Using these methods, no difficulties were encountered in assigning glutamic acid residues or in completely sequencing peptides that contained up to 25-30 residues. The relative ease with which the UP1 protein was sequenced, requiring only about a year to complete, and the comparatively modest amount of protein required, less than 5 mg, attests to the usefulness of water soluble carbodiimide coupling and solid-phase sequencing for determining the primary structures of proteins. In addition to serving as a basis for determining structural relationships among various mammalian single-stranded nucleic acid binding proteins, the amino acid sequence of UP1 reveals that the A1 hnRNP protein contains a region of internal sequence homology that apparently corresponds to two independent nucleic acid binding sites.     

The protein family of pyruvate:quinone oxidoreductases: Amino acid sequence conservation and taxonomic distribution

Pyruvate:quinone oxidoreductases (PQOs) catalyse the oxidative decarboxylation of pyruvate to acetate and concomitant reduction of quinone to quinol with the release of CO₂. They are thiamine pyrophosphate (TPP) and flavin-adenine dinucleotide (FAD) containing enzymes, which interact with the membrane in a monotopic way. PQOs are considered as part of alternatives to most recognized pyruvate catabolizing pathways, and little is known about their taxonomic distribution and structural/functional relationship.In this bioinformatics work we tackled these gaps in PQO knowledge. We used the KEGG database to identify PQO coding genes, performed a multiple sequence analysis which allowed us to study the amino acid conservation on these enzymes, and looked at their possible cellular function. We observed that PQOS are enzymes exclusively present in prokaryotes with most of the sequences identified in bacteria. Regarding the amino acid sequence conservation, we found that 75 amino acid residues (out of 570, on average) have a conservation over 90 %, and that the most conserved regions in the protein are observed around the TPP and FAD binding sites. We systematized the presence of conserved features involved in Mg²⁺, TPP and FAD binding, as well as residues directly linked to the catalytic mechanism. We also established the presence of a new motif named “HEH lock”, possibly involved in the dimerization process. The results here obtained for the PQO protein family contribute to a better understanding of the biochemistry of these respiratory enzymes.     

The amino acid sequence of a type I copper protein with an unusual serine- and hydroxyproline-rich C-terminal domain isolated from cucumber peelings.

We have determined the amino acid sequence of a small copper protein isolated from cucumber peelings. This cupredoxin contains 137 amino acids including a pyroglutamate as the first residue. The N-terminal 110 amino acid-long domain shows 30-37% identity to 2 other cupredoxins, stellacyanin and cucumber basic blue protein. A unique feature of this protein is a 27 amino acid-long C-terminal domain rich in 4-hydroxyproline and serine and resembling certain plant cell wall proteins. The prolines in this domain are hydroxylated to a different extent depending on the surrounding sequence.     

Amino acid sequence of the gene 0.3 protein of bacteriophage T7 and nucleotide sequence of its mRNA

The amino acid sequence of purified gene 0.3 protein of T7, the protein responsible for overcoming host restriction, has been determined. The nucleotide sequence of the 0.3 RNA, the messenger RNA that codes for both the 0.3 protein and the gene 0.4 protein, a T7 protein of unknown function, has also been determined. The 0.3 RNA is 578 nucleotides long, 509 of which are used to code for the 2 proteins. The coding sequences do not overlap, but the termination codon for the 0.3 protein and the presumed initiation codon for the 0.4 protein do overlap in the sequence UAAUG. The 0.3 protein is very acidic: 34 of its 116 amino acids are aspartic or glutamic acid and only 6 are arginine or lysine. The 0.3 protein contains no cysteine. The nucleotide sequence predicts that the 0.4 protein consists of 50 amino acids and contains no histidine or proline. The effects of different mutations indicate that a protein which contains only the first 87 amino acids of the 0.3 protein is unable to prevent host restriction in vivo; one that contains te first 93 amino acids has weak function; and one that has the first 94 amino acids (plus 2 that are not in the wild type sequence) is fully able to prevent host restriction. The apparently critical 94th amino acid is tryptophan. The mutant 0.3 proteins that contain 87 or more amino acids appear to be reasonably stable in vivo, but those that contain 78 or fewer are apparently too unstable to have been observed by gel electrophoresis.     

Identification and structural characterization of an unusual RING-like sequence within an extracellular biomineralization protein, AP7

The RING or Really Interesting New Gene represents a family of eukaryotic sequences that bind Zn (II) ions and participate in intracellular processes involving protein-protein interaction. Although found in over 400 different proteins, very little is known regarding the structure-function properties of these domains because of the aggregation problems associated with RING sequences. To augment this data set, we report an unusual 36 AA C-terminal sequence of an extracellular matrix mollusk shell protein, AP7, that exhibits partial homology to the RING family. This Cys, His-containing sequence, termed AP7C, binds Zn (II) and other multivalent ions, but does not utilize a tetracoordinate complexation scheme for binding such as that found in Zn (II) finger polypeptides. Moreover, unlike Zn (II) finger and RING domains, this 36 AA can fold into a relatively stable structure in the absence of Zn (II). This folded structure consists of three short helical segments (A, B, and C), with segments A and B separated by a 4 AA type I beta-turn region and segments B and C separated by a 7 AA loop-like region. Interestingly, the putative RING-like region, -RRPFHECALCYSI-, experiences slow conformational exchange between two structural states in solution, most likely in response to imido ring interconversion at P8 and P21. Poisson-Boltzmann solvation calculations reveal that the AP7C molecular surface possesses a cationic region near its N-terminus, which lies adjacent to the 30 AA mineral modification domain in the AP7 protein. Given that the AP7C sequence does not influence mineralization, it is probable that this cationic pseudo-RING region is utilized by the AP7 protein for other tasks such as protein-protein interaction within the mollusk shell matrix.     

Nucleotide sequence and derived amino acid sequence of a cDNA encoding human muscle carbonic anhydrase

We report the nucleotide (nt) sequence of a full length cDNA clone, pCA15, which encodes the human muscle-specific carbonic anhydrase, CAIII. pCA15 identifies a 1.7-kb mRNA, which is present at high levels in skeletal muscle, at much lower levels in cardiac and smooth muscle and which appears to be developmentally regulated. The CAIII mRNA is distinguished by a 887-nt long 3'-untranslated region, containing two AAUAAA signal sequences and is longer than either of the mRNAs encoding the erythrocyte CAs, CAI and CAII, which each have relatively shorter 3'-untranslated regions, 360 and 670 nt long, respectively. The derived amino acid (aa) sequence for human CAIII shows 85% homology with ox CAIII, 62% homology with human CAII and 54% with human CAI when simple pairwise aa comparisons are made. We describe an allelic variation at a TaqI restriction site for CAIII which occurs at high frequency in the European population.