Sequence of the cysteinyl-containing peptides of 4-aminobutyrate aminotransferase. Identification of sulfhydryl residues involved in intersubunit linkage

Three cysteine-containing tryptic peptides were isolated and sequenced from mitochondrial 4-aminobutyrate aminotransferase using DABIA (4-dimethylaminoazobenzene-4-iodoacetamide) as specific labeling reagent for sulfhydryl groups. The enzyme is a dimer made up of two identical subunits, but four out of the six cysteinyl residues/dimer form disulfide bonds when treated with iodosobenzoate to yield inactive enzyme species. To identify the cysteinyl residues undergoing reversible oxidation/reduction, the S-DABIA-labeling patterns of the fully reduced (active) and fully oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced enzyme contained three labeled peptides. If the enzyme was treated with iodosobenzoate prior to reaction with DABIA and tryptic digestion, only one labeled peptide was detected and identified (peptide I), indicating that the two missing cysteinyl-containing peptides (peptides II, III) have been oxidized. The sulfhydryl groups undergoing oxidation/reduction were found to be intersubunit, based on SDS/polyacrylamide gel electrophoresis results. The loss of catalytic activity of 4-aminobutyrate aminotransferase by oxidation of sulfhydryl residues is related to constraints imposed at the subunit interface by the insertion of disulfide bonds.     

The subunits of rabbit-muscle phosphofructokinase. A search for sequence repetition.

Rabbit muscle phosphofructokinase uniformly carboxymethylated with iodo[2-14C]acetate consists of subunits with a molecular weight of 80 000 +/- 5000. The subunit polypeptide chain contains 16 and 52 residues respectively of cysteine and arginine and, contrary to previous results, peptide mapping experiments gave no indication that phosphofructokinase chains yield fewer than the expected numbers of cysteine and arginine containing peptides. To test further for the possible occurrence of repeat sequences within a single subunit chain, cysteine-containing peptides were isolated and sequenced from tryptic and thermolytic digests of s-[2-14C]carboxymethylated phosphofructokinase. In all, 15 different cysteine sequences (comprising a total of 104 residues) were identified, showing that not more than one of an expected 16 cysteine-containing sequences is repeated, and that the subunits of phosphofructokinase are of unique sequence along their entire length. The near quantitative isolation of several cysteine-containing peptides shows further that all subunits are of similar if not identical sequence.     

Isolation and characterization of polypeptide at the picomole level. Pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate.

Polypeptides coupled with dimethylaminoazobenzene isothiocyanate through their amino groups to form dimethylaminoazobenzenethiocarbamoyl- (DABTC-)peptides can be separated by reversed-phase high-pressure liquid chromatography and detected in the visible region (436 nm). As little as 1 ng (2 pmol) of a DABTC-pentapeptide can be identified against a stable base-line with the signal-to-noise ratio of 10. The DABTC-peptides can also be recovered from the column, and their N-terminal amino acids (obtained by direct treatment with aqueous acid) and amino acid compositions and sequences can be all analysed at the picomole level. The power of this method is demonstrated by the complete separation and characterization of model peptides, peptide hormones and peptides derived from enzymic fragmentation of proteins. This new technique should provide a sensitive and efficient tool for peptide analysis at the nanogram level.     

A new isotype sequence (V kappa 27) of the variable region of kappa-light chains from a mouse hybridoma-derived anti-(streptococcal group A polysaccharide) antibody containing an additional cysteine residue. Application of the dimethylaminoazobenzene isothiocyanate technique for the isolation of peptides.

The first complete sequence of the variable region of a kappa-light chain (V kappa) from a mouse anti-(streptococcal group A polysaccharide) antibody (immunoglobulin 7S34.1) is reported. Immunoglobulin 7S34.1 was isolated from the ascitic fluid of hybridoma 7S34.1 previously cloned in vitro. A newly developed technique for the isolation of peptides by using pre-column formation of peptide derivatives with dimethylaminoazobenzene isothiocyanate also served to complete the sequence. The sequence of the variable region of the kappa-light chain of immunoglobulin 7S34.1 defines a new mouse V kappa isotype (V kappa 27) and is the first mouse immunoglobulin light-chain variable region to be shown to have an extra cysteine residue at position 48.     

Fructose 1,6-diphosphate aldolase of Drosophila melanogaster: comparative sequence analyses of the cysteine-containing peptides.

Following tryptic digestion four cysteine-containing peptides per monomer have been isolated from fructose 1,6-diphosphate aldolase of Drosophila melanogaster. Sequence analyses of the peptides showed that three of the four cysteinyl residues appear to occur in homologous positions to three of the eight cysteines of rabbit muscle aldolase. Moreover they seem to be homologous also to three of the six sulfhydryl groups in sturgeon aldolase. The fourth cysteine-containing peptide of Drosophila aldolase has no homologous SH peptide either in the rabbit or in the sturgeon enzyme, but corresponds to another tryptic peptide in the rabbit aldolase. As deduced from homology all four SH peptides are localized in the buried region of the molecule. This conclusion is confirmed by the fact that all four cysteine-containing peptides have been isolated from the central cyanogen bromide fragment. Drosophila aldolase has no exposed thiol groups, thus demonstrating that these residues are not essential either in catalytic activity or for the stabilization of the three-dimensional structure.     

Disulphide bridges of the heavy chain of human immunoglobulin G2

Amino acid sequences around the disulphide bridges of the heavy chain of an immunoglobulin of the gamma2 subclass have been studied. The protein was digested with pepsin and the digest fractionated by Sephadex. Screening of the eluate by one-dimensional electrophoresis of oxidized and unoxidized samples was used as an assay and pools of fractions were prepared. Identification by diagonal electrophoresis of several inter- and intra-chain disulphide bridges was done on the pooled fractions. The inter-heavy-chain bridged peptide included four cystine residues. Comparison with proteins of other human subclasses indicated that the intrachain bridges identified are the bridges of the invariable section of gamma2 heavy chains. The amino acid sequence of one cysteic acid peptide that may have been derived from the variable part of the molecule was determined. Partial reduction followed by carboxymethylation with radioactive iodoacetate of two proteins of the gamma2 class showed a number of labelled peptides that could be identified as being related to the inter-chain bonded cystine residues.     

Studies on the structure of rabbit muscle aldolase: Amino acid sequence of cysteine-containing peptides

Five cysteine-containing peptides have been isolated in nearly stoichemometric yields from the tryptic digests of the NH₂^? and COOH-terminal BrCN peptides of rabhit muscle aldolase and their sequence determined. Peptides NS1, NS2, and NS3, from the NH₂-terminal part of the enzyme have the following sequences: NS1, Val-Asp-Pro-Cys-Ile-Gly-Gly-Val-Ile-Leu-Phe-His-Glu-Thr-Leu-Tyr-Gln-Lys; NS2, Cys-Val-Leu-Lys; NS3, Cys-Ala-Glu-Tyr-Lys. The two peptides isolated from the COOH-terminal region are: CS1, Ala-Leu-Ala-Asn-Ser-Leu-Ala-Cys-Gln-Gly-Lys and CS2, Cys-Pro-Leu-Leu-Trp-Pro-Lys-Ala-Leu-Thr-Phe-Ser-Tyr-Gly-Arg. The Lys-Ala bond in peptide CS2 was found to be resistant to tryptic hydrolysis. The results provide the basis for assigning the positions of cysteine residues in the polypeptide chain. Cys-72 in peptide NS1 and Cys-336 in peptide CS1 are the residues that form a disulfide bridge when the enzyme is inactivated by oxidation with an o-phenanthroline-Cu²⁺ complex; Cys-287 in peptide CS2 in one of the two exposed residues, while Cys-134 and Cys-149 in peptides NS2 and NS3, respectively, are buried in the native enzyme. All of eight cysteine-containing peptides of rabbit muscle aldolase have now been sequenced, and structural homology of the α and β subunits extended to these regions.     

Use of N-(7-dimethylamino-4-methylcoumarinyl)maleinimide for the detection of cysteine-containing peptides in peptide maps

A fluorescent reagent N-[7-dimethylamino-4-methylcoumarinyl]maleinimide (DACM), which reacts selectively with protein thiols, was used in the detection of cysteine-containing peptides in peptide maps. Direct staining of peptide maps of glutathione and tryptic digested α₁-antitrypsin resulted in the finding of one and four cysteine-containing spots, respectively. All other peptides could be visualized after the DACM staining, by the use of fluorescamine. Amino acid analysis of all peptides showed that only the DACM fluorescent spots contained cysteine residues.     

Reversible labeling of cysteine-containing peptides allows their specific chromatographic isolation for non-gel proteome studies

We report upon a novel procedure to specifically isolate cysteine-containing peptides from a complex peptide mixture. Cysteines are converted to hydrophobic residues by mixed disulfide formation with Ellman's reagent. Proteins are subsequently digested with trypsin and the generated peptide mixture is a first time fractionated by reverse-phase high-performance liquid chromatography. Cysteinyl-peptides are isolated out of each primary fraction by a reduction step followed by a secondary peptide separation on the same column, performed under identical conditions as for the primary separation. The reducing agent removes the covalently attached group from the cysteine side chain, making cysteine-peptides more hydrophilic and, thereby, such peptides can be specifically collected during the secondary separation and are finally used to identify their precursor proteins using automated liquid chromatography tandem mass spectrometry. We show that this procedure efficiently isolates cysteine-peptides, making the sample mixture less complex for further analysis. This method was applied for the analysis of the proteomes of human platelets and enriched human plasma. In both proteomes, a significant number of low abundance proteins were identified next to extremely abundant ones. A dynamic range for protein identification spanning 4-5 orders of magnitude is demonstrated.     

The amino acid sequence of Pseudomonas fluorescens azurin

The amino acid sequence of Pseudomonas fluorescens azurin has been determined. The protein consists of a single peptide chain of 128 residues. There is one intra-chain disulphide bridge. The sequence was determined by isolation of the soluble tryptic peptides, and by exhaustive examination of the products of chymotryptic and peptic digestion. The sequence has been confirmed by the purification and analysis of the seven fragments obtained by cyanogen bromide treatment of the protein.     

Amino acid sequence pattern in the regulatory peptides.

The essential properties of the primary structure of regulatory peptides, i.e. amino acid residues and their combinations, which are characteristic of the whole population of regulatory peptides, have been revealed using statistical methodology. These properties are as follows: increased content of certain residues (Gly, Pro, Phe, Arg, Tyr, Met and Trp) as well as an increased rate of occurrence of certain pairs of residue as compared with proteins, a random sequence of residues and "nonregulatory" peptides. By representing regulatory peptides as a sequence of hydrophobic (2 types) and hydrophilic (3 types) segments, the pattern for alternation of these segments in regulatory peptides has been determined. The segments were classified into 5 types according to the peculiarities of mutual localization of hydrophobic and hydrophilic residues within the primary structure of regulatory peptides. As compared with proteins, "nonregulatory" peptides and a random sequence of segments, regulatory peptides were characterized by an increased frequency of 4 particular pairs of segments among 12 theoretically possible pairs. These 4 pairs are fragments of the periodic segment sequence with periods of 4 segments. The revealed pattern indicates that there exists a general principle of the regulatory peptide primary structure organization and possibly a common type of the regulatory peptides flexible peptide chain folding at the ligand-receptor complex formation.     

Mapping and analysis of protein sulfhydryl groups by modification with 2-bromoacetamido-4-nitrophenol

A method for identifying cysteine-containing peptides in proteins is presented using 2-bromoacetamido-4-nitrophenol (BNP) to introduce an easily detectable probe. The formation of a covalent bond between the protein sulfhydryl group and the acetamido moiety of BNP introduces a chromophore with an absorbance maximum at 410 nm. The modified protein can then be cleaved with appropriate proteases and the resulting peptides separated by chromatographic methods. Monitoring the effluent at a single wavelength (405 nm) provides a rapid and simple method of detecting and isolating only those peptides which contain cysteine residue(s). The nitrophenol derivative is stable under conditions required for protease cleavage. The reagent is therefore useful for locating cysteine-containing peptides in protein digests and can be used to explore the accessibility of different cysteines under a variety of conditions. The ease of modification, specificity of reaction, product stability, and simple detection of modified peptides make BNP ideal for investigation of cysteine residues.     

Chemical studies on the cysteine and terminal peptides in tryptic digests of actin

1. On exhaustive digestion of carboxymethylated actin in 6m-urea solutions with carboxypeptidase A, 1 mole of phenylalanine was liberated/43000g. of protein. At a lower urea concentration and in the absence of urea, carboxymethyl-cysteine (CMCys) was also liberated. 2. Three cysteine-containing peptides were identified by the study of peptide ;maps' of tryptic digests of actin treated with thiol reagents. 3. The three peptides, each containing one residue of CMCys, were isolated from tryptic digests of carboxymethylated actin by ion-exchange chromatography. 4. One of these peptides was possibly the N-terminal peptide and contained about 17-18 residues; another was CMCys-Asp-Ile-Asp-Ile-Arg; the other, CMCys-Phe, was the C-terminal tryptic peptide. 5. The chemical evidence suggests that the actin molecule consists of a single polypeptide chain of molecular weight about 44000.     

The disulphide bridges in a cellobiohydrolase and an endoglucanase from Trichoderma reesei.

The positions of the disulphide bridges of the 1,4-beta-glucan cellobiohydrolase (CBH I) of the fungus Trichoderma reesei have been investigated. The results can be summarized as follows. (1) The enzyme contains 12 disulphide bridges and no free cysteine residues. (2) The location of six disulphide bridges have been determined experimentally. (3) The bonding patterns of the two disulphide bridges in the C-terminal region is suggested on the basis of internal homology. (4) The remaining four disulphide bridges are put into two groups, each containing four half-cystine residues where two are adjacent. (5) A repeating bonding pattern is observed along the peptide chain and a non-local disulphide bond with an unusually long separation distance links the N-terminal and the C-terminal region. (6) The disulphide-bonded CNBr peptides of a 1,4-beta-glucan glucanohydrolase (endoglucanase II) from T. reesei have been isolated and a disulphide bonding pattern is suggested on the basis of the sequence homology between the two enzymes.     

Rotavirus VP4 and VP7-Derived Synthetic Peptides as Potential Substrates of Protein Disulfide Isomerase Lead to Inhibition of Rotavirus Infection

Rotavirus infection of MA104 cells has been shown to be inhibited by cell membrane-impermeant thiol/disulfide exchange inhibitors and anti-PDI antibodies. To characterise the amino acid sequences of rotavirus structural proteins potentially mediating cell surface PDI?Csubstrate interactions, rotavirus-derived peptides from VP4 and VP7 (RRV) and VP7 (Wa), and their modified versions containing serine instead of cysteine were synthesized. Cysteine-containing VP7 peptides corresponding to residues 189?C210 or 243?C263 caused an infectivity inhibitory effect of about 64 and 85?%, respectively, when added to cells. Changing cysteine to serine significantly decreased the inhibitory effect. A cysteine-containing peptide corresponding to VP4 residues 200?C219 and its scrambled version reduced infectivity by 92 and 80?%, respectively. A cysteine to serine change in the original VP4 200?C219 peptide did not affect its inhibitory effect. Non-rotavirus related sequences containing cysteine residues efficiently inhibited rotavirus infectivity. Antibodies against VP7 residues 189?C210 or 243?C263 significantly inhibited rotavirus infectivity only after virus attachment to cells had occurred, whereas those against VP4 200?C219 peptide inhibited infectivity irrespective of whether virus or cell-attached virus was antibody-treated. A direct PDI?Cpeptide interaction was shown by ELISA for cysteine-containing VP7 and VP4 peptides. Virus?Ccell attachment was unaffected by the peptides inhibiting virus infectivity. The results showed that even though cysteine residues in the peptides tested are important in both virus infectivity inhibition and in vitro PDI?Cpeptide interaction, the accompanying amino acid sequence also plays some role. As a whole, our findings further support our hypothesis that cell surface PDI from MA104 cells might be contributing to rotavirus entry at a post-attachment step.     

The covalent structure of collagen. The chymotrypsin, trypsin and hydroxylamine peptides derived from alpha2-CB4 of calf-skin collagen.

The cyanogen-bromide-derived peptide alpha2-CB4 from calf skin collagen, consisting of 321 amino acid residues, has been fragmented in order to obtain peptides suitable for automated sequential analysis. Digestion with chymotrypsin liberated six unique peptides consisting of 12, 17, 19, 54, 63 and 156 amino acid residues. Treatment of alpha2-CB4 with hydroxylamine yielded four peptides with 24, 87, 96 and 114 residues. No unspecific cleavage by hydroxylamine was encountered. All of the trypsin-derived peptides of alpha2-CB4 were isolated and characterized by their amino acid compositions. Most of the peptides isolated were ordered along the peptide chain of alpha2-CB4. Ordering of the peptides was greatly assisted by the isolation of double peptides from the chymotrypsin, trypsin and hydroxylamine-derived peptide mixtures.     

Alternate Positions for the Sulphydryl Group in β-Lactoglobulin: the Significance for Sulphydryl Location in Other Proteins

EARLIER studies of the location of the single cysteine residue and the two disulphide bridges in bovine β-lactoglobulins A and B¹, for each of which the monomer is a single chain of 162 residues and 18,000 molecular weight^2,3, led to the conclusion that the sulphydryl group is at position 69 and that the disulphides bridge positions 123 to 160 and 57 to 70. These results were based on diagonal peptide studies⁴ and on the composition of peptides in which the sulphydryl group had been labelled with ¹⁴C-iodoacetamide, the disulphide bridges being left intact. Use was made of the partial amino-acid sequence given by Frank and Braunitzer⁵ and the reasonable assumption was made that the sulphydryl occurred in only one position. Subsequently, Shaw⁶ has shown that the sequence of Frank and Braunitzer⁵ showing Cys residues adjacent at positions 69 and 70 is incorrect and that they are separated by a glutamine, the sequence for positions 67 to 71 for the Bvariant being Ala.Cys.Gln.Cys.Leu. Autoradiography of the dansyl amino-acid derivatives formed during the sequence determination of this pentapeptide indicated that both residues 68 and 70 seemed to have been labelled and so we have given further consideration to the sulphydryl location. It has been found that although it does occur at 68, with 57 and 70 disulphide bridged, there is also an equal amount of protein present with the sulphydryl at 70, with 57 and 68 disulphide bridged. We discuss this additional finding here and the significance for the determination of the location of sulphydryl groups in other proteins.     

An immunochemical aid to sequence determination of proteins.

A specific radioimmunoassay for peptides has been developed using ¹²⁵I-labeled peptides and a double-antibody precipitation. Cross-reacting peptides are measured by inhibition of the binding of the labeled cyanogen bromide peptide to its antibody. The assay, which allows detection of picomole quantities, was used to monitor the purification of two overlapping tryptic peptides from a complex mixture of peptides. These were shown to contain a portion of the sequence of the radio-labeled cyanogen bromide peptide and a portion of the sequence of a cyanogen bromide peptide which follows in the polypeptide chain. The need to analyze many fractions in a digest in order to locate a desired peptide is thus avoided. The general suitability of this method for the purification of specific peptides from digestion mixtures of other large proteins is discussed.     

Engineering new peptidic inhibitors from a natural chymotrypsin inhibitor.

Three model peptides of different sizes (17-24 amino acid residues) mimicking the chymotrypsin inhibitor SCGI (a peptide of 35 amino acid residues) isolated from Schistocerca gregaria were designed and prepared by convergent peptide synthesis. Selective formation of disulphide bridges in the closing step was achieved without selective protection of cysteine residues. The natural pattern of the two disulphide bridges was determined by 2D homonuclear 1H NMR techniques. All three model peptides were characterized by amino acid analysis. MS and CD spectra. Preliminary results revealed that the two smaller model peptides exhibit no Inhibitory activity, whereas the larger one shows limited inhibition of chymotrypsin.     

Sticky egyptians: a technique for assembling genes encoding constrained peptides of variable length.

Naturally occurring peptides, such as those produced by the poisonous marine snails of the genus Conus , have the ability to form tight, highly specific molecular interactions. The rigidity of the peptide framework which promotes these interactions is usually maintained by disulphide bonds, and it seems that the overall main chain conformation (or fold) of the peptide is determined by its length and the sequence distribution of the pairs of cysteine residues participating in these bonds. The fold of the peptide in turn is largely responsible for its shape. Since highly effective molecular interactions occur between species complementary in shape, we reasoned that peptides with the greatest potential in therapy or diagnosis would be found in a library of shapes, those peptides with a shape complementary to a given target being identified, for example, by selection. As a first step towards constructing such a peptide shape library, we have developed a method for assembling DNA fragments which encode an even number of cysteine residues and which are of variable length. We describe this method here.