A hydrophobic tryptic peptide from bovine white matter proteolipid.

A hydrophobic, chloroform-soluble tryptic peptide with a molecular weight of approximately 4000 has been purified from the bovine white matter proteolipid protein. Its primary structure was obtained by a combination of solid-phase Edman degradation and mass spectrometry. A major part of the tryptic peptide appears to be inaccessible to the action of proteolytic enzymes. The peptide spans the three cyanogen bromide peptides located by Jollès et al. (Biochem. Biophys. Res. Commun. (1979) 87, 619--626) at the COOH-terminal region of the intact protein. Secondary structure calculations for this region indicate a segregation into discrete domains, with most of the tryptic peptide corresponding to a highly ordered, hydrophobic domain; an equal probability for alpha-helical or beta-structure is predicted for this region.

Multiple-copy genes: production and modification of monomeric peptides from large multimeric fusion proteins.

A vector system has been designed for obtaining high yields of polypeptides synthesized in Escherichia coli. Multiple copies of a synthetic gene encoding the neuropeptide substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) have been linked and fused to the lacZ gene. Each copy of the SP gene was flanked by codons for methionine to create sites for cleavage by cyanogen bromide (CNBr). The isolated multimeric SP fusion protein was converted to monomers of SP analog, each containing a carboxyl-terminal homoserine lactone (Hse-lactone) residue (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Hse-lactone), upon treatment with CNBr in formic acid. The Hse-lactone moiety was subjected to chemical modifications to produce an SP Hse amide. This method permits synthesis of peptide amide analogs and other peptide derivatives by combining recombinant DNA techniques and chemical methods.

Microscale elucidation of an N-linked glycosylation site by comparative high-performance liquid chromatographic peptide mapping.

The identification of specific sites of post-translational modifications of polypeptides is an important component in understanding protein structure-function relationships. This report describes the application of specific enzymatic methods of N-linked oligosaccharide removal (endo H and endo F) to the rapid microscale identification of specific sites of N-linked glycosylation. Following deglycosylation the protein is subjected to proteolytic digestion and comparative high-performance liquid chromatographic peptide mapping with an unmodified protein digest. Peptides from which oligosaccharide(s) have been removed exhibit a measurable increase in retention time on reversed-phase high-performance liquid chromatography and can be clearly identified. Once identified, the peptide can be subjected to direct protein microsequence analysis to elucidate the specific site of glycosylation. As illustrated here, these methods are compatible with microscale protein purification by sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by electroelution and are applicable to the 100-pmol range.

The amino acid sequence of elongation factor Tu of Escherichia coli. The large cyanogen bromide peptides.

The amino acid sequences of three large cyanogen bromide peptides, which comprise 289 of the 393 amino acids in Escherichia coli elongation factor Tu, have been determined. The peptides were digested with trypsin, Staphylococcus aureus protease, and chymotrypsin to give overlapping subfragments, which were separated by high performance liquid chromatography. Peptides were sequenced by solid-phase Edman degradation. Sequence analysis of these peptides completes the sequence of elongation factor Tu described in the companion article (Laursen, R. A., L'Italien, J. J., Nagarkatti, S. N., and Miller, D. L. (1981) J. Biol. Chem. 256, 8102-8109).

Primary structure of the hydrophobic plant protein crambin.

Crambin, a hydrophobic plant seed protein, consists of a single chain of 46 amino acids with a calculated molecular weight of 4720. The primary structure was determined by using solid-phase sequencing techniques and was confirmed through X-ray crystallographic analysis of the protein at 1.5-A resolution [Hendrickson, W. A., & Teeter, M. M. (1981) Nature (London) 290, 107-112]. High-performance liquid chromatographic separation of the proteolytic fragments from crambin led to the identification of two sites of microheterogeneity. The three disulfide bonds were located at positions 3-40, 4-32, and 16-26 from the crystallographic data. Comparison of the primary structure with known sequences revealed that crambin is homologous with the plant toxins purothionin and viscotoxin. Methods to estimate protein secondary structure were applied and found to predict all of crambin's structure except its amphiphilic helix.

The amino acid sequence of elongation factor Tu of Escherichia coli. The complete sequence.

The complete amino acid sequence of elongation factor Tu of Escherichia coli has been established by sequencing overlapping cyanogen bromide and tryptic peptides. Sequence analysis of peptides was done primarily by solid-phase Edman degradation. Elongation factor Tu is a single chain polypeptide composed of 393 amino acids (Mr = 43,225). Its NH2 terminus is blocked with an acetyl group, as determined by mass spectroscopy, and lysine 56 is partially methylated. The cysteine residues associated with aminoacyl tRNA and guanosine nucleotide binding are located at positions 81 and 137, respectively. Although elongation factor Tu is coded for by two genes, the only site of microheterogeneity found was at the carboxyl terminus (residue 393), which is either glycine or serine. Comparison of the first 140 amino acids of elongation factor Tu and of elongation factor G shows a strong (31%) sequence homology. In addition, secondary structure calculations predict remarkable conformational similarities between the two proteins. The NH2-terminal region of elongation factor Tu appears to be composed of two beta-sheet domains connected by an exposed, alpha-helical bridge, which includes a 14-amino acid segment released by limited treatment with trypsin. Structural features of the aminoacyl-tRNA binding site are discussed in the light of sequence and other chemical and biochemical data.

Streptococcal pyrogenic exotoxin type A (scarlet fever toxin) is related to Staphylococcus aureus enterotoxin B.

The nucleotide sequence of the gene encoding group A streptococcal pyrogenic exotoxin type A (SPE A) was determined by the dideoxy chain termination method. The first 30 residues of the translation product represented a hydrophobic signal peptide. The mature protein was 220 amino acids in length and had a molecular weight of 25,805. It has significant protein sequence homology with Staphylococcus aureus enterotoxin B but not with other proteins in the Dayhoff library.

Characterization of the Escherichia coli SSB-113 mutant single-stranded DNA-binding protein. Cloning of the gene, DNA and protein sequence analysis, high pressure liquid chromatography peptide mapping, and DNA-binding studies.

The ssb-113 (formerly lexC113) gene encoding a mutant single-stranded DNA binding protein (SSB) has been cloned into plasmid pSC101 resulting in 5- to 10-fold more mutant protein than strains carrying only one (chromosomal) copy of the gene. Analysis of tryptic and chymotryptic peptides of the mutant protein by high pressure liquid chromatography and solid phase protein sequencing has shown that the ssb-113 mutation results in the substitution of serine for proline at residue 176 of SSB. This change could only occur in one step by a C leads to T transition in the DNA sequence. Physicochemical studies of the homogeneous mutant protein have shown that it binds as well as wild type SSB to single-stranded DNA and that it is a slightly better helix-destabilizing protein than wild type SSB as measured by its ability to lower the thermal melting transition of poly[d(A-T)]. In vivo studies of ssb-113 strains carrying the cloned ssb-113 gene in pSC101 have shown that overproduction of the mutant protein does not complement the temperature-sensitive conditional lethality caused by the ssb-113 mutation when present in single gene copy in contrast to effects recently observed in ssb-1 strains overproducing the ssb-1 encoded protein (Chase, J. W., Murphy, J. B., Whittier, R. F., Lorensen, E., and Sninsky, J. J. (1983) J. Mol. Biol. 164, 193-211). Also noted in this report are two corrections to the DNA sequence of wild type SSB, one of which places glycine (codon GGC) at residue 133 rather than serine as previously reported (Sancar, A., Williams, K. R., Chase, J. W., and Rupp, W. D. (1981) Proc. Natl. Acad. Sci. U.S.A. 78, 4274-4278). The second correction to the DNA sequence is in the serine 39 codon, previously reported to be TCA and now correctly shown to be TCC.

High-level expression in Escherichia coli of biologically active bovine growth hormone.

High-level synthesis of bovine growth hormone (bGH) in Escherichia coli was achieved by maximizing gene transcription and optimizing the translational efficiency of bGH mRNA. Nearly all of the recombinant hormone was found in the pellet fraction after bacterial cell lysis. This property allowed the purification of bGH nearly to homogeneity. Protein sequence analysis indicated that greater than 93% of the purified hormone had the amino-terminal methionine residue removed by E. coli, yielding mature bGH. In a hypophysectomized rat assay system, purified bacterial-produced bGH demonstrated growth-promoting activity equivalent to that of pituitary-derived bovine growth hormone.

Trypanosoma congolense: structure and molecular organization of the surface glycoproteins of two early bloodstream variants.

The complete primary structures of two variant specific glycoproteins (VSGs) of the nannomonad Trypanosoma (N.) congolense are presented. These coat proteins subserve the function of antigenic variation. The secondary structure potentials of both VSGs have been calculated. The amino acid sequences and secondary structure potentials of these VSGs have been compared with the primary structures and secondary structure potentials of several Trypanosoma brucei complex VSGs. In homologous regions, the T. brucei complex VSGs show a pattern of sharply contrasting secondary structure potentials. It has been suggested previously that this pattern gives rise to different folding structures in different members of this polygene protein family. Thus, different short regions of the polypeptide sequence are exposed as antigenic "caps" on the solvent-exposed surface of intact trypanosomes. A sharply contrasting secondary structure potential pattern is also found in regions of the two T. congolense VSGs. However, there is little homology of primary structure between each of the two T. congolense VSGs and any member of the T. brucei complex VSG polygene family whose primary structure has been determined.