Arrangement of proteins O-8 and O-9 in outer membrane of Escherichia coli K-12. Existence of homotrimers and heterotrimers.

1. The molecular arrangement of major outer membrane proteins O-8 and O-9 that exist as trimers has been studied by means of cross-linking with dimethylsuberimidate. 2. The cross-linked samples were examined on a urea/sodium dodecyl sulfate/polyacrylamide gel which was developed to separate cross-linked trimer and dimer of O-8 from those of O-9. 3. Cells simultaneously synthesizing both O-8 and O-9 formed heterotrimers (trimers containing both proteins) as well as homotrimers. 4. Quantitative analyses revealed that there was no discrimination between O-8 and O-9 in the assembly process to form trimers. 5. When cells were grown sequentially under two different sets of conditions so that the cells synthesized either one of the two proteins in the first stage and the other in the second stage of growth, no heterotrimers were formed. This result indicates that subunit exchange did not take place between trimers which had been incorporated into the outer membrane.     

Influence of molecular size and osmolarity of sugars and dextrans on the synthesis of outer membrane proteins O-8 and O-9 of Escherichia coli K-12.

Supplementation of the growth medium with high concentrations of sugars or low-molecular-weight dextrans results in a drastic change in the ratio of outer membrane proteins O-8 and O-9, due to induction of O-8 synthesis and suppression of O-9 synthesis. Sugars and dextrans of molecular weights greater than 600 to 700 switched the synthesis of O-9 to that of O-8 more effectively than those of lower molecular weight, although the effect was almost the same within each of the two groups irrespective of the differences in molecular weight within the group. Proteins O-8 or O-9, or both, are responsible for the formation of pores that allow the passive diffusion of hydrophilic molecules whose molecular weights are smaller than about 600 (T. Nakae, Biochem. Biophys. Res. Commun. 71:877-884, 1976). The results indicate that substances that cannot pass through the outer membrane switch the synthesis of O-9 to that of O-8 more effectively than those that can penetrate this membrane with the aid of O-8, O-9, or both. It is suggested that the osmotic pressure exerted on the outer membrane plays an important role in the regulation of synthesis of the two proteins.     

Interactions of outer membrane proteins O-8 and O-9 with peptidoglycan sacculus of Escherichia coli K-12.

The outer membrane proteins O-8 and O-9 were specifically bound to the peptidoglycan sacculus in sodium dodecyl sulfate (SDS) solution. Other cellular proteins failed to interact with the peptidoglycan sacculus under the same conditions. When the outer membrane was preheated in SDS solution, the binding did not take place. Optimum binding was observed at pH 8 in the presence of 5 mM Mg2+. A high concentration of sodium chloride strongly inhibited the binding. The effects of these factors on the bindings of O-8 and O-9 required neither the bound nor the free form of Braun's lipoprotein, nor was the binding of either protein necessary for the binding of the other. Proteins O-8 and O-9 were also found in the peptidoglycan sacculus when it was prepared from cells in SDS solution at 60 degrees. A dilution experiment showed that the complex was not an artifact. The mode of interaction between these proteins and peptidoglycan in the preparation was similar to that in the reassembled O-8-O-9-peptidoglycan complex, as judged from the sensitivity to sodium chloride and temperature. The physiological importance of the complex is discussed in relation to the assembly of the outer membrane on the cell surface.     

Luteinizing hormone of the sperm whale. Isolation, separation into subunits and study of the amino acid sequence of the alpha-subunit

The luteinizing hormone isolated from sperm-whale pituitary was separated into two subunits, alpha- and beta-, by ion-exchange chromatography on sulfoethyl-Sephadex. The hormone subunits were reconstituted, carboxymethylated and cleaved by BrCN and proteolytic enzymes. In order to block tryptic hydrolysis at lysine residues the alpha-subunit was subjected to maleylation. Large-sized fragments of BrCN were cleaved by chymotrypsin and trypsin, while large-sized fragments of trypsin were split by chymotrypsin. The resulting peptides were separated by gel filtration on Sephadex, ion-exchange chromatography on Aminex A-5 and thin-layer partition chromatography on cellulose. The amino acid sequence of the peptides was determined by the Edman method, using identification of the N-terminal amino acids in a reaction with dansyl chloride or dimethylaminoazobenzene-4-isothiocyanate. It was shown that the alpha-subunit of the luteinizing hormone is a peptide chain consisting of 96 amino acid residues with covalently linked carbon chains at asparagine residues at positions 56 and 82. The N-terminal amino acid of the alpha-subunit is phenylalanine, the C-terminal amino acid is serine. The alpha-subunit is heterogeneous at the N-end, i. e. beside phenylalanine it contains threonine and trace amounts of proline, aspartate, glutamate and glycine.     

Combinations of turns in proteins.

We observed that beta- and gamma-turns in protein structure may be associated as peptides representing combinations of turns that span between nine and 26 amino acid residues along the polypeptide backbone chain and often correspond to loops in the protein structure. Around 475 peptides resulted from the analysis of a non-redundant data set corresponding to 248 protein crystal structures selected from the Protein Data Bank. Nearly 40% protein chains are associated with two or more peptides and the peptides with nine and 10 amino acid residues are more frequent. A maximum of four distinct peptides varying in number of amino acid residues were observed in at least 10 proteins along the same protein chain. Nearly 80% peptides comprise type IV beta-turns that are associated with irregular dihedral angle values suggesting this may be important for the conformational diversity associated with the loops in proteins. In general, predominant interactions that possibly stabilize these peptides involve main-chain and side-chain interactions with solvent, in addition to hydrogen bond, salt-bridge and non-bonded interactions. Majority of the peptides were observed in hydrolase, oxidoreductase, transferase, serine proteinase/inhibitor complex, electron transport/electron transfer and lyase proteins.     

Isolation and characterization of the cyanogen bromide fragments of lobster arginine kinase (homarus vulgaris).

Arginine kinase was aminoethylated in order to block the five free thiol groups on the native enzyme, and then submitted to BrCN cleavage. The BrCN resulting peptides were soluble in propionic acid (10 percent) and subsequently submitted to gel-filtration. The large polypeptide subfractions were citraconylated and resubmitted to differnt gelchromatographies, whereas the short peptide subfractions were submitted to preparative paper electrochromatographies. Eight peptides of 2, 11, 17, 25, 61, 82, 86 and 132 amino acid residues were isolated, one of which is the overlapping of two peptides. The amino acid composition and the end group of all the isolated peptides were established. The short peptides (2, 11 and 17 residues) were sequenced. All peptides possess homoserine at C-terminal position because one methionyl residue is situated at the C-terminal position in the native protein. The polypeptide with 132 residues possessed N-acetylated residue at N-terminal position: therefore this polypeptide is located at the N-terminal position in the protein. The sum and account of each amino acid of the seven isolated peptides were compared to those of the intact protein: the sum of the seven peptides is 331 amino acid residues, whereas the whole protein contains 342 residues. The molecular weight of arginine kinase is revised and calculated on the basis of the present results (37, 687).     

Purification and primary structure of novel lipid transfer proteins from germinated lentil (<Emphasis Type="Italic">Lens culinaris</Emphasis>) seeds

A subfamily of eight novel lipid transfer proteins designated as Lc-LTP1-8 was found in the lentil Lens culinaris. Lc-LTP2, Lc-LTP4, Lc-LTP7, and Lc-LTP8 were purified from germinated lentil seeds, and their molecular masses (9268.7, 9282.7, 9121.5, 9135.5 daltons) and complete amino acid sequences were determined. The purified proteins consist of 92-93 amino acid residues, have four disulfide bonds, and inhibit growth of Agrobacterium tumefaciens. Total RNA was isolated from germinated lentil seeds, RT-PCR and cloning were performed, and the cDNAs of six LTPs were sequenced. Precursor 116-118-residue proteins with 24-25-residue signal peptides were found, and two of them are purified proteins Lc-LTP2 and Lc-LTP4.     

Serratia protease. Amino acid sequences of both termini, the 53 residues in the middle region containing the sole methionine residue, and a probable zinc-binding region

Reexamination of the molecular mass and the amino acid composition of Serratia protease revealed the presence of 1 mol of methionine per mol of protein (about 46K daltons), and this was confirmed by BrCN cleavage followed by separation of the two fragments. The sole methionine residue was located near the middle region of the molecule. The amino(N)-terminal sequence was determined by Edman degradation of the protein and studies of several proteolytic peptides, establishing a sequence of 18 residues with a heterogeneous N-terminus. The carboxyl(C)-terminal sequence was determined by carboxypeptidase A digestion and tritium-labeling of the citraconylated C-terminal half segment to be -Phe-Ile-Val. The sequences of a total of 53 residues containing the methionine residue and a total of 38 residues containing two histidine residues were established by the application of various conventional methods to a BrCN peptide and several proteolytic peptides. The segment containing the histidine residues was homologous with that containing the two histidine residues chelating the zinc atom of thermolysin. The 38-residue segment may be directly connected to the 53-residue segment.     

Primary structure of a base non-specific and adenylic acid preferential ribonuclease from the fruit bodies of Lentinus edodes.

The complete primary structure of a base non-specific and adenylic acid preferential RNase (RNase Le2) from the fruit bodies of Lentinus edodes was analyzed. The sequence was mostly determined by analysis of the peptides generated by V8 protease digestion and BrCN cleavage (including alpha-chymotryptic, and V8 protease digest of BrCN fragments). It consists of 239 amino acid residues. The molecular weight is 25831. The location of 10 half cystine residues were almost superimposable on those of known fungal RNases of the RNase T2 family. The sequence homologies between RNase Le2 and four known fungal RNases of the RNase T2 family, RNase T2, RNase M, RNase Trv, and RNase Rh, are 102, 103, 109, and 74, respectively. The homologous sequences are concentrated around the three histidines, which are supposed to form the active site of RNase T2 family RNases.     

Strain specificity of outer membrane proteins in Escherichia coli.

Outer membrane proteins of various strains of Escherichia coli were compared using three different systems of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The outer membranes of E. coli K-12, E. coli B, and E. coli J-5 had distinctive protein compositions. As regards proteins which interact with peptidoglycan, E. coli K-12 contained O-8 and O-9, while E. coli B possessed one protein which migrated to the position of O-9. Although E. coli J-5 possessed two such proteins, O-8' and O-9', their positions on polyacrylamide gel were different from those of O-8 and O-9. Protein O-7, which migrates slightly more slowly than O-8, was found specifically in E. coli K-12. Proteins O-10 and O-11 were found in all strains tested, although the relative amounts were different depending on the strain. Strains of E. coli K-12 and E. coli J-5 gave three major bands, O-2a, O-2b, and O-3, in the region of high molecular weight. These proteins were repressed by iron in the cultivation media. Strains of E. coli B, on the other hand, gave only O-2b and O-3. E. coli J-5 gave two other major bands in this region, but the amounts were not controlled by iron in the cultivation media.     

Tetrahymena HMG nonhistone chromosomal protein. Isolation and amino acid sequence lacking the N- and C-terminal domains of vertebrate HMG 1

The complete amino acid sequence of a high mobility group (HMG) nonhistone chromosomal protein of the ciliated protozoan Tetrahymena pyriformis (GL strain) was determined. This protein was extracted with 0.5 M HClO4 together with histone H1 (molar ratio 1:1) from the whole histone extract, then purified by gel filtration and reverse-phase HPLC. The HMG protein showed a single electrophoretic band on SDS gel electrophoresis. The amino acid sequence was determined by Edman degradation of intact protein, BrCN fragments, and their staphylococcal protease and tryptic peptides. Thus the total sequence, consisting of 99 amino acid residues and having a molecular weight of 11,626, was completely determined. Phosphorus analysis of the tryptic peptides, containing serine or threonine, showed that this HMG protein was phosphorylated at two positions, each 6-7%, and contained 0.15 mol phosphate/mol protein. This Tetrahymena HMG is rather similar to the central part of vertebrate HMG 1 in terms of the amino acid sequence and the hydropathy profile.     

Outer membrane proteins of Escherichia coli. V. Evidence that protein 1 and bacteriophage-directed protein 2 are different polypeptides.

Protein 1 from the outer membrane of Escherichia coli K-12 and protein 2 from a phage PA-2 lysogen of the same strain were isolated by differential sodium dodecyl sulfate extraction and purified by ion-exchange and gel filtration chromatography. Rabbit antisera were prepared against these proteins and showed no cross-reaction between proteins 1 and 2. The proteins have the same N-terminal amino acid but show small yet significant differences in amino acid composition. The proteins were cleaved with cyanogenbromide in solvents containing both formic acid and trifluoroacetic acid. By comparing the cleavage in these solvents, it was established that protein 1 yielded 5 cyanogen bromide peptides, and the sum of the molecular weights of these was equivalent to the molecular weight of the uncleaved protein. Protein 2 yielded 4 cyanogen bromide peptides, none of which was identical to those of protein 1, and the sum of these peptides was also equivalent to the apparent molecular weight of the uncleaved protein. Significant differences were also observed when tryptic peptides from the two proteins were compared. These results indicate that protein 1 and the phage-directed protein 2 are distinct, different, and apparently homogeneous proteins.     

Selective oxidation of methionine residues in proteins.

Methionine residues in peptides and proteins were oxidized to methionine sulfoxides by mild oxidizing reagents such as chloramine-T and N-chlorosuccinimide at neutral and slightly alkaline pH. With chloramine-T cysteine was also oxidized to cystine but no other amino acid was modified; with N-chlorosuccinimide tryptophans were oxidized as well. In peptides and denaturated proteins all methionine residues were quantitatively oxidized, while in native proteins only exposed methionine residues could be modified. Extent of oxidation of methionine residues was determined by quantitative modification of the unoxidized methionine residues with cyanogen bromide (while methionine sulfoxide residues remained intact), followed by acid hydrolysis and amino acid analysis. Methionine was determined as homoserine and methionine sulfoxide was reduced back to methionine. Sites of oxidation were identified in a similar way by cleaving the unoxidized methionyl peptide bonds with cyanogen bromide, followed by quantitative end-group analysis of the new amino-terminal amino acids (by an automatic sequencer).     

DNA and histone H1 interact with different domains of HMG 1 and 2 proteins.

High mobility group (HMG) proteins 1 and 2 from calf thymus have been digested under structuring conditions (0.35 M NaCl, pH 7.1) with two proteases of different specificities, trypsin and V8. The two proteases give a different but restricted pattern of peptides in a time course digestion study. However, when the interactions of the peptides with DNA are studied by blotting, a closely related peptide from HMG-1 and -2 does not show any apparent binding. This peptide, from the V8 protease digestion, has been isolated by DNA-cellulose chromatography and has the amino acid composition predicted for a fragment containing the two C-terminal domains of the protein, i.e., approximately residues 74-243 for HMG-1. The same peptide shows the only interaction detectable with labelled histone H1. A separate function for the different domains of HMG proteins 1 and 2 is proposed.     

Structure of an antifreeze polypeptide from the sea raven. Disulfide bonds and similarity to lectin-binding proteins.

The antifreeze polypeptide (AFP) from the sea raven, Hemitripterus americanus, is a member of the cystine-rich class of blood antifreeze proteins which enable survival of certain fishes at sub-zero temperatures. Sea raven AFP contains 129 residues with 10 half-cystine residues. We have analyzed these half-cystine residues and established that all 10 of the half-cystine residues appeared to be involved in disulfide bond formation and that disulfide bonds linked Cys7 to Cys18, Cys35 to Cys125, and Cys89 to Cys117. These assignments were established by extensive proteolytic digestions of native AFP using pepsin and thermolysin and purification of the peptides by Sephadex G-15 gel filtration chromatography, anion exchange chromatography, and C18 reverse-phase high performance liquid chromatography. Cystine-containing peptides were detected by a colorimetric assay using nitrothiosulfobenzoate. Disulfide-containing peptides were reduced and alkylated, purified, and analyzed by amino acid analysis. The unreduced disulfide-linked peptides were sequenced directly by automated Edman degradations to confirm the disulfide assignments. Possible arrangements of the two remaining disulfide bonds include linkages Cys69/111 to Cys100/101. The sea raven AFP shares structural similarity with pancreatic stone protein and several lectin-binding proteins, especially with respect to half-cystines, glycines, and bulky aromatic residues. Two of the disulfide linkages we determined for sea raven AFP: Cys7-Cys18 and Cys35-Cys125, are conserved in these proteins. These similarities in covalent structure suggest that the sea raven AFP, pancreatic stone protein, and several lectin-binding proteins comprise a family of proteins which may possess a common fold.     

Structural studies on wheat (Triticum aestivum) proteins lacking phenylalanine and histidine residues.

1. Three very similar proteins, each of approx. 120 amino acid residues but lacking phenylalanine and histidine, were isolated from wheat (Triticum aestivum) flour in sufficient quantities for further structural studies. 2. Each protein, after reduction and carboxymethylation, was cleaved at the three methionine residues with CNBr to give four major peptides, which were isolated. These peptides are suitable for future sequencing studies, as the sums of their amino acid compositions are in good agreement with those of the whole proteins. 3. The N- and C-terminal peptides were identified. 4. Evidence from amino acid analyses, N-terminal amino acids and electrophoretic mobilities of the peptides suggests a high degree of homology between the proteins. Definite differences in C-terminal amino acids and the number of glycine, alanine and arginine residues were found in the C-terminal peptides.     

Primary structure of a base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi

The complete primary structure of a base non-specific and adenylic acid preferential RNase (RNase M) from Aspergillus saitoi was determined. The sequence was determined by analysis of the peptides generated by digestion of heat-denatured RNase M with lysylendopeptidase, and the peptides generated from RCM RNase M by digestion with staphylococcal V8 protease or chemical cleavage with BrCN. It consisted of 238 amino acid residues and carbohydrate moiety attached to the 74th asparagine residue. The molecular weight of the protein moiety deduced from the sequence was 26,596. The locations of 10 half cystine residues are almost superimposable on those of RNase Rh from Rhizopus niveus and RNase T2 from Aspergillus oryzae which have similar base specificity. The homology between RNase M and RNase Rh and RNase T2 amounted to 97 and 160 amino acid residues, respectively. The amino acid sequences conserved in the three RNases are concentrated around the three histidine residues, which are supposed to form part of the active sites of these RNases.     

根癌土壤杆菌C58 Cereon中分泌蛋白信号肽分析

利用SignalP3．0、LipoP1．0、TMHMM2．0和TargetP1．014种蛋白分析软件预测了Agrobacterium tumefaciens C58 Cereon菌株全部基因组的4554个ORF编码的蛋白信号肽,共发现203个信号肽,且它们的氨基酸残基相对保守。其中158条具分泌型信号肽,9条具RR-motif型信号肽,28条具信号肽酶Ⅱ型信号肽,8条具细菌素-信息素型信号肽,但只有分泌蛋白AGR-C-1878p和AGR-C-1880p的信号肽氨基酸残基完全相同,表明信号肽是高度变异的。     

The requirement of a positive charge at the amino terminus can be compensated for by a longer central hydrophobic stretch in the functioning of signal peptides.

A variety of model presecretory proteins, proOmpF-Lpps, possessing different numbers of lysine residues (0, 2, and 4) as positively charged amino acid residues and different numbers of leucine residues (7, 8, and 9) as hydrophobic amino acid residues in their signal peptides were constructed. The effect of positive charges on the in vitro translocation efficiency markedly differed with the number of leucine residues. Positive charges were strongly required for translocation when the hydrophobic region comprised 7 or 8 leucine residues, whereas the translocation of proOmpF-Lpps possessing 9 leucine residues took place efficiently even in the absence of positive charges and the introduction of positive charges did not significantly enhance the translocation efficiency. The translocation of all the proOmpF-Lpps, including one possessing no positive charge, was ATP-, protonmotive force-, and SecA-dependent and accompanied by signal peptide cleavage, indicating that they are translocated via the usual secretory pathway. It is likely that the requirement of positive charges can be compensated for by a longer hydrophobic stretch in the functioning of the signal peptide.     

Tetrahymena histone H1. Isolation and amino acid sequence lacking the central hydrophobic domain conserved in other H1 histones

The complete amino acid sequence of a single H1 histone of the protozoan Tetrahymena pyriformis was determined, following previous determinations of the sequences of histones H2B, H2A, H3, and H4. Only a single H1 species was obtained by fractionation of a 0.5 M HClO4-soluble fraction from the whole histone extract and further purification. This starting material for sequencing contained 1.1 mol/mol phosphate and showed a single electrophoretic band after dephosphorylation. The sequence determination was performed by Edman degradation of BrCN fragments, staphylococcal protease peptides, and tryptic peptides, as well as secondary peptides from one BrCN fragment and one staphylococcal protease peptide. Phosphorus analysis of the tryptic peptides, containing serine or threonine, showed that five sites of the sequence were phosphorylated to various extents (5-30%). Thus, the total sequence, consisting of 165 amino acid residues and having a molecular weight of 17,942 in the unmodified form, was completely determined. This unusually small H1 sequence differs substantially from the human spleen H1 sequence of 218 residues, having larger proportions of hydrophilic residues and smaller proportions of hydrophobic residues. Comparison of the distribution pattern of hydrophilic and hydrophobic residues, between the protozoan and human sequences, showed that the protozoan sequence lacks the central hydrophobic domain that is conserved in the known vertebrate and other H1 histones. The implications for the function of H1 are discussed from the evolutionary viewpoint.