Primary structures of and genes for new ribosomal proteins A and B in Escherichia coli

We determined the partial primary structures of and identified the genes for new basic proteins A and B in Escherichia coli ribosomal 50S subunits, found by means of an improved two-dimensional gel electrophoresis method. The sequence up to the 17th amino acid of protein B was in agreement with that of the X gene in the spc operon. The gene for protein A was searched for in the GenBank data base using the sequence up to the 35th amino acid, and was found at a locus between infC and rplT. The base sequence indicated that protein A contained 64 amino acids and had a molecular weight of 6,984. We conclude that proteins A and B are intrinsic ribosomal proteins, and propose calling their genes, rpmI and rpmJ, respectively.     

Directed evolution of highly homologous proteins with different folds by phage display: implications for the protein folding code

To better understand how amino acid sequences specify unique tertiary folds, we have used random mutagenesis and phage display selection to evolve proteins with a high degree of sequence identity but different tertiary structures (homologous heteromorphs). The starting proteins in this evolutionary process were the IgG binding domains of streptococcal protein G (G(B)) and staphylococcal protein A (A(B)). These nonhomologous domains are similar in size and function but have different folds. G(B) has an alpha/beta fold, and A(B) is a three-helix bundle (3-alpha). IgG binding function is used to select for mutant proteins which retain the correct tertiary structure as the level of sequence identity is increased. A detailed thermodynamic analysis of the folding reactions and binding reactions for a pair of homologous heteromorphs (59% identical) is presented. High-resolution NMR structures of the pair are presented by He et al. [(2005) Biochemistry 44, 14055-14061]. Because the homologous but heteromorphic proteins are identical at most positions in their sequence, their essential folding signals must reside in the positions of nonidentity. Further, the thermodynamic linkage between folding and binding is used to assess the propensity of one sequence to adopt two unique folds.     

The nucleotide sequences of the ponA and ponB genes encoding penicillin-binding protein 1A and 1B of Escherichia coli K12

Penicillin-binding proteins 1A and 1B of Escherichia coli are the major peptidoglycan transglycosylase-transpeptidases that catalyse the polymerisation and insertion of peptidoglycan precursors into the bacterial cell wall during cell elongation. The nucleotide sequence of a 2764-base-pair fragment of DNA that contained the ponA gene, encoding penicillin-binding protein 1A, was determined. The sequence predicted that penicillin-binding protein 1A had a relative molecular mass of 93 500 (850 amino acids). The amino-terminus of the protein had the features of a signal peptide but it is not known if this peptide is removed during insertion of the protein into the cytoplasmic membrane. The nucleotide sequence of a 2758-base-pair fragment of DNA that contained the ponB gene, encoding penicillin-binding protein 1B, was also determined. Penicillin-binding protein 1B consists of two major components which were shown to result from the use of alternative sites for the initiation of translation. The large and small forms of penicillin-binding protein 1B were predicted to have relative molecular masses of 94 100 and 88 800 (844 and 799 amino acids). The amino acid sequences of penicillin-binding proteins 1A and 1B could be aligned if two large gaps were introduced into the latter sequence and the two proteins then showed about 30% identity. The amino acid sequences of the proteins showed no extensive similarity to the sequences of penicillin-binding proteins 3 or 5, or to the class A or class C beta-lactamases. Two short regions of amino acid similarity were, however, found between penicillin-binding proteins 1A and 1B and the other penicillin-binding proteins and beta-lactamases. One of these included the predicted active-site serine residue which was located towards the middle of the sequences of penicillin-binding proteins 1A, 1B and 3, within the conserved sequence Gly-Ser-Xaa-Xaa-Lys-Pro. The other region was 19-40 residues to the amino-terminal side of the active-site serine and may be part of a conserved penicillin-binding site in these proteins.     

Complete amino acid sequence of protein B

The complete amino acid sequence of protein B (= CAMP factor) of Streptococcus agalactiae has been determined. The sequence data were obtained mainly by manual sequencing of peptides derived from digestion with lysyl-peptidase, clostripain and Staphylococcus aureus protease and by solid phase sequencing of cyanogen bromide fragments. The protein contains 226 amino acids and has an Mr of 25,263. The sequence was compared with sequences of other Fc-binding proteins and partial sequence homology was found between protein B and the Fc-binding region of protein A.     

Melanoma antigens as modified normal gene sequences

Melanomas are highly aggressive tumors with a well-documented antigenic nature. Several melanoma antigens have been reported, four of which, p97, Ia-like antigen, B700, and A have been implicated as having regions in common with normally occurring proteins. P97 has partial sequence homology with transferrin and lactotransferrin, Ia-like antigen is immunologically cross-reactive with alpha and beta chains of Ia-like proteins, A is a variant of alpha actin, and B700 resembles a normal melanosomal membrane protein. In addition, B700 has partial sequence homology to serum albumins. These observations suggest that melanoma tumors can produce antigenic proteins by modification of normally occurring proteins. The possible mechanisms are discussed.     

The SR splicing factors ASF/SF2 and SC35 have antagonistic effects on intronic enhancer-dependent splicing of the beta-tropomyosin alternative exon 6A.

Exons 6A and 6B of the chicken beta-tropomyosin gene are mutually exclusive and selected in a tissue-specific manner. Exon 6A is present in non-muscle and smooth muscle cells, while exon 6B is present in skeletal muscle cells. In this study we have investigated the mechanism underlying exon 6A recognition in non-muscle cells. Previous reports have identified a pyrimidine-rich intronic enhancer sequence (S4) downstream of exon 6A as essential for exon 6A 5'-splice site recognition. We show here that preincubation of HeLa cell extracts with an excess of RNA containing this sequence specifically inhibits exon 6A recognition by the splicing machinery. Splicing inhibition by an excess of this RNA can be rescued by addition of the SR protein ASF/SF2, but not by the SR proteins SC35 or 9G8. ASF/SF2 stimulates exon 6A splicing through specific interaction with the enhancer sequence. Surprisingly, SC35 behaves as an inhibitor of exon 6A splicing, since addition to HeLa nuclear extracts of increasing amounts of the SC35 protein completely abolish the stimulatory effect of ASF/SF2 on exon 6A splicing. We conclude that exon 6A recognition in vitro depends on the ratio of the ASF/SF2 to SC35 SR proteins. Taken together our results suggest that variations in the level or activity of these proteins could contribute to the tissue-specific choice of beta-tropomyosin exon 6A. In support of this we show that SR proteins isolated from skeletal muscle tissues are less efficient for exon 6A stimulation than SR proteins isolated from HeLa cells.     

Using Common Spatial Distributions of Atoms to Relate Functionally Divergent Influenza Virus N10 and N11 Protein Structures to Functionally Characterized Neuraminidase Structures,Toxin Cell Entry Domains,and Non-Influenza Virus Cell Entry Domains

The ability to identify the functional correlates of structural and sequence variation in proteins is a critical capability. We related structures of influenza A N10 and N11 proteins that have no established function to structures of proteins with known function by identifying spatially conserved atoms. We identified atoms with common distributed spatial occupancy in PDB structures of N10 protein, N11 protein, an influenza A neuraminidase, an influenza B neuraminidase, and a bacterial neuraminidase. By superposing these spatially conserved atoms, we aligned the structures and associated molecules. We report spatially and sequence invariant residues in the aligned structures. Spatially invariant residues in the N6 and influenza B neuraminidase active sites were found in previously unidentified spatially equivalent sites in the N10 and N11 proteins. We found the corresponding secondary and tertiary structures of the aligned proteins to be largely identical despite significant sequence divergence. We found structural precedent in known non-neuraminidase structures for residues exhibiting structural and sequence divergence in the aligned structures. In N10 protein, we identified staphylococcal enterotoxin I-like domains. In N11 protein, we identified hepatitis E E2S-like domains, SARS spike protein-like domains, and toxin components shared by alpha-bungarotoxin, staphylococcal enterotoxin I, anthrax lethal factor, clostridium botulinum neurotoxin, and clostridium tetanus toxin. The presence of active site components common to the N6, influenza B, and S. pneumoniae neuraminidases in the N10 and N11 proteins, combined with the absence of apparent neuraminidase function, suggests that the role of neuraminidases in H17N10 and H18N11 emerging influenza A viruses may have changed. The presentation of E2S-like, SARS spike protein-like, or toxin-like domains by the N10 and N11 proteins in these emerging viruses may indicate that H17N10 and H18N11 sialidase-facilitated cell entry has been supplemented or replaced by sialidase-independent receptor binding to an expanded cell population that may include neurons and T-cells.     

Monoclonal antibody specific to a subclass of polyproline-Arg motif provides evidence for the presence of an snRNA-free spliceosomal Sm protein complex in vivo: implications for molecular interactions involving proline-rich sequences of Sm B/B' proteins.

The human spliceosomal Sm B/B' proteins are essential for the biogenesis of the snRNP particles. B/B' proteins contain several clusters of the PPPPGM/IR sequence, which occurs within the C-terminus of Sm B/B'. This sequence is very similar to the PPPPPGHR sequence of the cytoplasmic tail of the CD2 receptor and closely resembles the class II of SH3 ligands, suggesting a similarly important role. We report that a monoclonal antibody (3E10) against the PPPPPGHR sequence recognizes spliceosomal Sm B/B' proteins. Proteins that are specifically immunoprecipitated by 3E10 include Sm B, B', D1, D2, D3, E, F, and G. However, unlike Y12 and other anti-Sm immunoprecipitates, 3E10 immunoprecipitates appear to lack the U1 snRNP-specific proteins A and C and U snRNAs. These findings indicate that 3E10 recognizes a subset of Sm protein core and suggest the presence of snRNA-free Sm protein complex(es) in vivo. We propose that the epitope binding for 3E10 may become unaccessible upon interactions of Sm proteins and their subsequent incorporation into the core particles. The Sm proline-rich sequences may have an important role in mediating protein-protein interactions necessary for the proper snRNP core assembly or function, or both. To our knowledge, 3E10 is the first well characterized mAb specific for a subclass of polyproline-arg motif recognizing Sm B/B' and CD2 proteins. 3E10 antibody can be used to further characterize the nature of protein components in the snRNA-free Sm subcore protein complex(es) that are formed during the snRNP core assembly steps.     

Lamins A, B and C share an epitope with the common domain of intermediate filament proteins

Peripheral lamina were isolated from rat liver nuclei and probed with a monoclonal antibody reactive against a common domain of all intermediate filament (IF) proteins. The antibody reacted against lamins A and C as expected from their known sequence homology with IF proteins. Lamin B was also recognized by the antibody. These results indicate that lamin B, despite important biochemical differences vs lamins A and C, also shares sequence homology with IF proteins.     

Mutational analysis of the R64 oriT region: requirement for precise location of the NikA-binding sequence.

Conjugative DNA transfer of IncI1 plasmid R64 is initiated by the introduction of a site- and strand-specific nick into the origin of transfer (oriT). In R64 oriT, 17-bp (repeat A and B) and 8-bp inverted-repeat sequences with mismatches are located 8 bp away from the nick site. The nicking is mediated by R64 NikA and NikB proteins. To analyze the functional organization of the R64 oriT region, various deletion, insertion, and substitution mutations were introduced into a 92-bp minimal R64 oriT sequence and their effects on oriT function were investigated. This detailed analysis confirms our previous prediction that the R64 oriT region consists of an oriT core sequence and additional sequences necessary for full oriT activity. The oriT core sequence consists of the repeat A sequence, which is recognized by R64 NikA protein, and the nick region sequence, which is conserved among various origins of transfer and is most probably recognized by NikB protein. The oriT core sequence is sufficient for NikAB-mediated oriT-specific nicking. Furthermore, it was shown that the repeat A sequence is essential for localization to a precise position relative to the nick site for oriT function. This seems to be required for the formation of a functional ternary complex consisting of NikA and NikB proteins and oriT DNA. The repeat B sequence and 8-bp inverted repeat sequences are suggested to be required for the termination of DNA transfer.     

A protein factor binding to an octamer motif in the gamma-globin promoter disappears upon induction of differentiation and hemoglobin synthesis in K562 cells.

Using the electrophoretic mobility shift assay and the footprinting technique, we studied the binding of nuclear proteins from erythroid and non erythroid human cells to the promoter region of the human gamma-globin gene. Two regions (A and B) of the promoter are bound by proteins present in uninduced K562 cells, but not in induced K562 cells nor in fetal liver erythroblasts; a protein binding to region A is also present in a variety of lymphoid and myeloid cells. Region B is centered on an octamer sequence identical to that present in immunoglobulin promoter and enhancers and other eukaryotic promoters; a B region binding protein common to K562 and other cells efficiently binds the octamer containing region of the histone H2B gene, while different B region proteins are more specific for uninduced K562 cells and the gamma-globin octamer containing fragment. The possible role of these nuclear proteins in gamma-globin gene regulation and/or cell differentiation is discussed.     

Glycinin A3B4 mRNA. Cloning and sequencing of double-stranded cDNA complementary to a soybean storage protein

The cDNA clones encoding the precursor form of glycinin A3B4 subunit have been identified from a library of soybean cotyledonary cDNA clones in the plasmid pBR322 by a combination of differential colony hybridizations, and then by immunoprecipitation of hybrid-selected translation product with A3-mono-specific antiserum. A recombinant plasmid, designated pGA3B41425, from one of six clones covering codons for the NH2-terminal region of the subunit was sequenced, and the amino acid sequence was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 516 amino acids. Analysis of this cDNA also showed that it contained 1786 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 46 nucleotides, a signal peptide region corresponding to 24 amino acids, an A3 acidic subunit region corresponding to 320 amino acids followed by a B4 basic subunit region corresponding to 172 amino acids, and a 3'-terminal nontranslated region of 192 nucleotides, which contained two characteristic AAUAAA sequences that ended 110 nucleotides and 26 nucleotides from a 3'-terminal poly(A) segment, respectively. Our results confirm that glycinin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs via disulfide bonds. The inferred amino acid sequence of the mature basic subunit, B4, was compared to that of the basic subunit of pea legumin, Leg Beta, which contained 185 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall 42% of the amino acid positions are identical in both proteins. These results led us to conclude that both storage proteins have a common ancestor.     

Proteomic analysis of Lyme disease: global protein comparison of three strains of Borrelia burgdorferi

The Borrelia burgdorferi spirochete is the causative agent of Lyme disease, the most common tick-borne disease in the United States. It has been studied extensively to help understand its pathogenicity of infection and how it can persist in different mammalian hosts. We report the proteomic analysis of the archetype B. burgdorferi B31 strain and two other strains (ND40, and JD-1) having different Borrelia pathotypes using strong cation exchange fractionation of proteolytic peptides followed by high-resolution, reversed phase capillary liquid chromatography coupled with ion trap tandem mass spectrometric analysis. Protein identification was facilitated by the availability of the complete B31 genome sequence. A total of 665 Borrelia proteins were identified representing approximately 38% coverage of the theoretical B31 proteome. A significant overlap was observed between the identified proteins in direct comparisons between any two strains (>72%), but distinct differences were observed among identified hypothetical and outer membrane proteins of the three strains. Such a concurrent proteomic overview of three Borrelia strains based upon only the B31 genome sequence is shown to provide significant insights into the presence or absence of specific proteins and a broad overall comparison among strains.     

Homologous sequences in cholera toxin A and B subunits to peptide domains in myelin basic protein

Recent reports that myelin basic protein (MBP) can be ADP-ribosylated and contains specific sites that bind GTP and GM1 ganglioside, have suggested an analogy to the properties of cholera toxin. Comparisons of pairs of sequences between these two proteins yielded two regions of homology between MBP and the cholera toxin B (chol B) subunit, and one region of homology with the cholera toxin A (chol A) subunit. The matching sites within chol B consisted of a 17 amino acid residue sequence (residues 30-46 in chol B and residues 102-118 in human-MBP, hMBP, p less than 0.0007) and an 11 residue span (residues 31-41 in chol B and sequence 29-39 in hMBP, p less than 0.0004). The homologous site within chol A corresponded to an 11 residue span (residues 130-140 in chol A and 67-77 in hMBP sequence, p less than 0.00007). Since portions of the cholera toxin sequence are virtually identical to sections of the sequence in E. coli toxin, the homology is also valid for the same sequences in this toxin. The highly antigenic behavior of MBP that is related to the induction of experimental allergic encephalomyelitis may be paralleled by comparable neural pathology from the homologous regions of cholera toxin.