Comprising Amino Acid Residues Research Articles

Truncated forms of Escherichia coli lactose permease: models for study of biosynthesis and membrane insertion.

Using in vitro DNA manipulations, we constructed different lacY alleles encoding mutant proteins of the Escherichia coli lactose carrier. With respect to structural models developed for lactose permease, the truncated polypeptides represent model systems containing approximately one, two, four, and five of the N-terminal membrane-spanning alpha-helices. In addition, a protein carrying a deletion of predicted helices 3 and 4 was obtained. The different proteins were radiolabeled in plasmid-bearing E. coli minicells and were found to be stably integrated into the lipid bilayer. The truncated polypeptides of 50, 71, 143, and 174 N-terminal amino acid residues resembled the wild-type protein in their solubilization characteristics, whereas the mutant protein carrying an internal deletion of amino acid residues 72 to 142 of the lactose carrier behaved differently. Minicell membrane vesicles containing truncated proteins comprising amino acid residues 1 to 143 or 1 to 174 were subjected to limited proteolysis. Upon digestion with proteases of different specificities, the same characteristic fragment that was also produced from the membrane-associated wild-type protein was found to accumulate under these conditions. It has previously been shown to contain the intact N terminus of lactose permease. This supports the idea of an independent folding and membrane insertion of this segment even in the absence of the C-terminal part of the molecule. The results suggest that the N-terminal region of the lactose permease represents a well-defined structural domain.

Polypeptide sequences involved in the cleavage of DNA by the restriction endonuclease EcoRI.

We have prepared a variety of fragments of the restriction endonuclease EcoRI by partial or total CNBr or acid cleavage of the protein. These fragments were isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. They were analyzed in a qualitative manner for phosphodiesterase activity. Antibodies against these fragments were elicited in rats and tested for binding to native EcoRI in an enzyme-linked immunoassay. We conclude from these experiments that the DNA binding site of EcoRI is located in the COOH-terminal half of the molecule, close to and probably comprising amino acid residues 137 to 157. This conclusion is reinforced by the observation that this sequence shows homology to the sequences of the recognition helix of other gene-regulatory proteins.

Identification of an antigenic determinant within the phylogenetically conserved triprolyl region of myelin basic protein.

Synthetic peptide SP6 (RTPPPSG), comprising amino acid residues 98-103-Gly of myelin basic protein (MBP), and a series of peptide analogs were used to probe the structural requirements for antigenicity of a highly conserved region of a self protein. By means of a liquid-phase radioimmunoassay, antibody responses directed toward this determinant in both multi-specific anti-MBP and monospecific anti-peptide antisera were measured. The specificities of the antibodies present in the anti-MBP and anti-peptide antisera were examined by an equilibrium competitive inhibition radioimmunoassay by using the set of related peptides, as well as intact MBP from different species. Although the fine specificities of the reagent antisera differed, competitive inhibition analyses with intact MBP revealed a cross-reactive determinant involving residues 99-100 (Thr-Pro). This suggests that the neighborhood of the triprolyl region of MBP, despite its strong phylogenetic conservation, serves as an immunogen for humoral responses whether presented as a hapten-carrier conjugate or in the context of intact MBP. The latter supports the contention that the general antigenicity of a protein need not require sequence differences between the immunizing protein and its counterpart in the host.

Common features of the yes and src gene products defined by peptide-specific antibodies.

Anti-peptide antibodies generated against a hydrophilic domain of pp60src comprising amino acid residues 498 through 512 were shown to be cross-reactive with the corresponding region in the yes transforming proteins encoded by Yamaguchi 73 and Esh sarcoma viruses. This cross-reactivity was demonstrated by immunoblot and immunoprecipitation analyses, and the identity of the proteins was verified by partial proteolytic mapping. By utilizing a combination of immunofluorescence and interference-reflection microscopy, these cross-reactive anti-peptide antibodies were shown to produce an immunofluorescence staining pattern in Yamaguchi 73 and Esh sarcoma virus-transformed chicken embryo fibroblasts remarkably similar to that pp60src in Rous sarcoma virus-infected chicken cells. Like the src gene products, the yes transformation-specific polyproteins were found to be concentrated within adhesion plaque structures and needle-like interdigitating cell-cell junctions. This analogous subcellular distribution suggests that these onc proteins are functionally related and may share common intracellular targets.

A colicin A fragment containing the receptor binding domain can be directed to the periplasmic space in E. coli through gene fusion

The central region of the colicin A polypeptide chain has been fused to the N-terminal part of β-lactamase through genetic recombination. This region comprising amino acid residues 70–335 confers on the hybrid protein the ability to protect sensitive cells from the lethal action of colicin A. Although colicin A belongs to the cytoplasmic compartment of E. coli, export of the hybrid protein to the periplasmic space was promoted by the signal peptide of β-lactamase.

A 61,000-dalton truncated large T-antigen is uniformly expressed in hamster cells transformed by polyomavirus.

Various polyomavirus-transformed hamster cell lines derived from tumors or from infected hamster cell cultures synthesized polyoma middle and small tumor (T)-antigens but no full-size large T-antigen. Instead, all cell lines produced the same or similar polyoma T-antigen-related proteins of ca. 61 kilodaltons (kDal). Like large T-antigen synthesized in lytically infected mouse cells, the 61-kDal proteins were phosphoproteins showing electrophoretic and charge heterogeneities. Chromatographic analysis of the methionine-containing tryptic peptides indicated that the 61-kDal proteins were truncated forms of large T-antigen comprising amino acid residues 1 to 485 (+/- 25). Analysis of viral DNA present in hamster chromosomal DNA of three independently isolated cell lines confirmed that synthesis of the 61-kDal proteins was due to a discontinuity in the large T-antigen coding sequence, most likely located between 7 and 8.9 map units on the polyoma DNA map. The three cell lines yielded essentially the same patterns of viral DNA-containing restriction enzyme fragments, suggesting that insertion of viral DNA into the hamster chromosomes took place at closely similar sites.

Primary structure of the polypeptide chain elongation factor Tu from E. coli. I. Amino acid sequence of fragment B.

The complete amino acid sequence of Fragment B obtained by the limited tryptic digestion of E. coli polypeptide chain elongation factor Tu (EF-Tu) was determined. Seven peptides formed from Fragment B by cleavage with cyanogen bromide (designated as CB1 to CB7 according to their order of alignment from N- to C-termini of Fragment B) were purified, and six of them were completely sequenced by the manual method of sequential Edman degradation with direct identification of the phenylthiohydantoin-amino acids. The remaining one cyanogen bromide peptide (CB6) containing 109 amino acid residues was further digested with trypsin. Twelve tryptic peptides (designated as T1 to T12 according to their order of alignment from N- to C-termini of CB6) were isolated, and their amino acid sequences were analyzed. The alignment of CB peptides was based on the results of the automated sequence analysis of Fragment B from its N-terminal, and the sequence analysis of the overlapping peptides containing sulfhydryl groups obtained by the complete tryptic digestion of Fragment B. The alignment of peptides T1 to T12 on CB6 was based on the result of the automated sequence analysis of CB6, and the sequence of the overlapping peptide obtained by the chemical cleavage of CB6 at the tryptophan residue using cyanogen bromide in heptafluorobutyric acid. The nucleotide sequence of the tuf A gene was also utilized for the alignment of these peptides. Fragment B comprises amino acid residues 59 to 263 of E. coli EF-Tu, which consists of 393 amino acids. It contains two functional (SH1 and SH2) and one non-functional (SH3) sulfhydryl groups of EF-Tu. All of the five histidine residues in Fragment B were distributed within the first N-terminal quarter, and three of them were found to be clustered around SH2. Although E. coli EF-Tu consists of two gene products (tuf A and tuf B), no microheterogeneity was found in the amino acid sequence of Fragment B.