Adjacent Cysteine Research Articles

Variants of ribonuclease inhibitor that resist oxidation.

Human ribonuclease inhibitor (hRI) is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonucleases. hRI has 32 cysteine residues. The oxidation of these cysteine residues to form disulfide bonds is a rapid, cooperative process that inactivates hRI. The most proximal cysteine residues in native hRI are two pairs that are adjacent in sequence: Cys94 and Cys95, and Cys328 and Cys329. A cystine formed from such adjacent cysteine residues would likely contain a perturbing cis peptide bond within its eight-membered ring, which would disrupt the structure of hRI and could facilitate further oxidation. We find that replacing Cys328 and Cys329 with alanine residues has little effect on the affinity of hRI for bovine pancreatic ribonuclease A (RNase A), but increases its resistance to oxidation by 10- to 15-fold. Similar effects are observed for the single variants, C328A hRI and C329A hRI, suggesting that oxidation resistance arises from the inability to form a Cys328-Cys329 disulfide bond. Replacing Cys94 and Cys95 with alanine residues increases oxidation resistance to a lesser extent, and decreases the affinity of hRI for RNase A. The C328A, C329A, and C328A/C329A variants are likely to be more useful than wild-type hRI for inhibiting pancreatic-type ribonucleases in vitro and in vivo. We conclude that replacing adjacent cysteine residues can confer oxidation resistance in a protein.

NMR study of crosslinking by oxidation of four-cysteine polypeptide models of the elastic network phase of wool fibre

An elastic network of the matrix phase of wool fibres is formed by disulphide bonds between cysteine residues of the high-sulphur (HS) proteins of the matrix. To elucidate the pattern of the disulphide cross linking we applied 1H and 15N NMR spectroscopy to synthetic fragments of the HS proteins and also to the products of their oxidation by air. Two different regimes, oxidation in solution and oxidation in bulk, were employed. In solution of the four-Cys-residue peptides, the sequential dipeptide Cys-Cys repeat (present two times in model peptide 1) and the pentapeptide Cys-Xxx-Pro-Yyy-Cys repeat (present in model peptide 2) were found to form intrarepeat disulphide bonds and not to form a network. In bulk the behaviour of the two repeats differs dramatically: the peptide with the Cys-Xxx-Pro-Yyy-Cys oxidizes to a network, but for the peptide with the Cys-Cys repeat the oxidation in bulk does not differ from that in solution and, therefore, does not lead to network formation. The latter is the consequence of an exceptional propensity of the two adjacent cysteines of the dipeptide repeat to form a small intrarepeat cyclocystine loop which is much stronger than that of any other pair of the cysteines of the system. The formation of the cyclocystine loops effectively eliminates the potential of the Cys residues of the dipeptide repeat to serve as cross links of a network. A special structural function of the Cys-Cys repeat and its implications for the elasticity of the network of the matrix are discussed. © 1998 Society of Chemical Industry.

Molecular cloning of feline CC–chemokine cDNAs

cDNA clones of feline chemokines, MIP-1 α, MIP-1 β and RANTES, were molecularly isolated with the purpose of using these sequences for future investigation of the inhibitory effects on lentivirus entry and their role in immunological functions. The feline MIP-1 α and MIP-1 β cDNA clones spanned their entire coding regions encoding 93 and 92 amino acids, respectively. The amino acid sequences of feline MIP-1 α and MIP-1 β compared to those of their human, mouse and rat counterparts showed similarities of 75.3–79.6% and 73.9–88.0%, respectively. Feline MIP-1 α and MIP-1 β had four conserved cysteines with a structure made up of the first two cysteines that are characteristic of the CC–chemokine subfamily. The amino terminal of these MIP-1 α and MIP-1 β sequences was distinctly hydrophobic, suggesting that they may function as signal peptides. A partial cDNA clone consisting of 193 bp was obtained for feline RANTES, and it also showed a high degree of sequence similarity to those of other species and contained the characteristic structure made up of adjacent cysteines. These molecular clones of feline chemokines will be useful in the examination of their inhibitory effect on the cellular entry of feline immunodeficiency virus.

Disulfide Mass Mapping in Proteins Containing Adjacent Cysteines Is Possible with Cyanylation/Cleavage Methodology

Disulfide Mass Mapping in Proteins Containing Adjacent Cysteines Is Possible with Cyanylation/Cleavage Methodology

Disulphide Bonds of Adjacent Cysteine Residues in Low Molecular Weight Subunits of Wheat Glutenin

The adjacent cysteine residues Cf1and Cf2located in the homologous sequence division III of LMW subunits of glutenin, and of α- and γ-gliadins are important elements of the disulphide structure of these proteins. Two different analytical approaches were used to assign the disulphide linkages of the -Cys-Cys- sequence found in four cystine-containing peptides derived from LMW subunits of glutenin. Direct Edman degradation of the acetylated and cyanogen bromide treated peptide with detection of di-PTH-cystine was one method. Partial reduction of peptides with Tris(2-carboxyethyl)-phosphine hydrochloride, subsequent separation of fragments by RP-HPLC, alkylation with iodoacetamide and sequence analysis was the second method. Both these methodologies gave identical results. It was shown that the first of the adjacent cysteines (Cf1) is linked with another cysteine residue of division III (Cc) and that the second one of the pair (Cf2) is linked with a cysteine residue in the C-terminal division V (Cy) of the primary structure. A model for the complete disulphide structure of LMW subunits is presented.

The structure and function of the PQQ-containing quinoprotein dehydrogenases.

Bacterial methanol and glucose dehydrogenases containing a novel type of prosthetic group, subsequently identified as pyrrolo-quinoline quinone (PQQ), were first described about 30 years ago. Quinoproteins were originally defined as proteins containing PQQ but this definition has since been broadened to include those proteins containing other types of quinone-containing prosthetic groups, and the X-ray structures of representatives of each type of quinoprotein have recently been published. This review is mainly concerned with the structure and function of the PQQ-containing methanol dehydrogenase, whose structure has been determined at high resolution, and related proteins. Their basic structure consists of a 'propeller' fold superbarrel made up of 8-sheet 'propeller blades' which are held together by novel tryptophan-docking motifs. In methanol dehydrogenase the PQQ in the active site is coordinated to a Ca2+ ion and is maintained in position by a stacked tryptophan and a novel 8-membered ring structure made up of a disulphide bridge between adjacent cysteine residues. This review describes these features and discusses them in relation to previously proposed mechanisms for this enzyme.

Novel roles for Fe-S clusters in stabilizing or generating radical intermediates

Abstract

Inhibition of human mitochondrial aldehyde dehydrogenase by the disulfiram metabolite S-methyl- N, N-diethylthiocarbamoyl sulfoxide : Structural characterization of the enzyme adduct by HPLC-tandem mass spectrometry

S-Methyl- N, N-diethylthiocarbamoyl sulfoxide (MeDTC-SO) is a known metabolite of the aversion therapy drug disulfiram (DSF). MeDTC-SO is also a potent inhibitor of human mitochondrial aldehyde dehydrogenase (hmALDH) with an ic 50 of 1.5 μM. Inhibition of the enzyme by MeDTC-SO resulted in the addition of ~100 Da to the molecular mass of the intact protein, as determined by on-line HPLC-electrospray ionization MS (LC-MS). Dialysis of the inhibited protein did not reverse the inhibition, and the molecular mass of 54,533 Da (± 0.01%) remained unchanged, indicating that a covalent modification of the protein had occurred. Proteolytic digestion of hmALDH under basic conditions using trypsin at pH 7.8 revealed that the adduct was base labile. However, treating the adducted protein with endopeptidase-Glu-C at pH 3.7 produced a peptide adduct at MH + = 4924, tentatively attributable to a carbamoylated peptide. This peptide contains three adjacent cysteines, one of which has been implicated as a key amino acid in the highly conserved active site region of ALDH. A pepsin digestion of hmALDH carried out at pH 3.7 and subsequent LC-MS analysis revealed an ion at MH + = 501.5, corresponding to the carbamoylated peptide FNQGQC 1C 2C 3. This peptide contains the same adjacent active site cysteines. This latter peptide was subjected to LC-MS/MS, which enabled us to determine that the site of carbamoylation was at Cys 2. The MS/MS product ion data also confirmed the presence of a carbamoyl group as the adduct species.

Conversion of Desulforedoxin into a Rubredoxin Center

Rubredoxin and desulforedoxin both contain an Fe(S-Cys)4center. However the spectroscopic properties of the center in desulforedoxin differ from rubredoxin. These differences arise from a distortion of the metal site hypothesized to result from adjacent cysteine residues in the primary sequence of desulforedoxin. Two desulforedoxin mutants were generated in which either a G or P-V were inserted between adjacent cysteines. Both mutants exhibited optical spectra with maxima at 278, 345, 380, 480, and 560 nm while the low temperature X-band EPR spectra indicated high-spin Fe3+ions with large rhombic distortions (E/D = 0.21-0.23). These spectroscopic properties are distinct from wild type desulforedoxin and virtually identical to rubredoxin.

A novel methodology for assignment of disulfide bond pairings in proteins.

A novel methodology is described for the assignment of disulfide bonds in proteins of known sequence. The denatured protein is subjected to limited reduction by tris(2-carboxyethyl)phosphine (TCEP) in pH 3.0 citrate buffer to produce a mixture of partially reduced protein isomers; the nascent sulfhydryls are immediately cyanylated by 1-cyano-4-dimethylamino-pyridinium tetrafluoroborate (CDAP) under the same buffered conditions. The cyanylated protein isomers, separated by and collected from reversed-phase HPLC, are subjected to cleavage of the peptide bonds on the N-terminal side of cyanylated cysteines in aqueous ammonia to form truncated peptides that are still linked by residual disulfide bonds. The remaining disulfide bonds are then completely reduced to give a mixture of peptides that can be mass mapped by MALDI-MS. The masses of the resulting peptide fragments are related to the location of the paired cysteines that had undergone reduction, cyanylation, and cleavage. A side reaction, beta-elimination, often accompanies cleavage and produces overlapped peptides that provide complementary confirmation for the assignment. This strategy minimizes disulfide bond scrambling and is simple, fast, and sensitive. The feasibility of the new approach is demonstrated in the analysis of model proteins that contain various disulfide bond linkages, including adjacent cysteines. Experimental conditions are optimized for protein partial reduction, sulfhydryl cyanylation, and chemical cleavage reactions.

Identification of Two Palmitoyl Groups in Octopus Rhodopsin

We determined the structure and site of fatty acid incorporated in octopus rhodopsin using a combination of fluorescence label and enzymatic cleavage methods in conjunction with fast-atom bombardment (FAB) mass spectrometry. A single peptide containing two adjacent cysteines, Cys337 and Cys338, was successfully isolated using the fluorescence from a dye conjugated to Cys345. The FAB mass spectrometric analysis of the peptide (323Phe-340Phe) revealed that two palmitoyl groups are linked to Cys337 and Cys338 via thioester bonds in octopus rhodopsin as in bovine rhodopsin.

Cloning and sequence analysis of the candidate nicotinic acetylcholine receptor alpha subunit gene tar-1 from Trichostrongylus colubriformis

A T. colubriformis genomic library in lambda EMBL3 was screened for sequences homologous to the Caenorhabditis elegans unc-38 nicotinic acetylcholine receptor (nAChR) α-subunit gene. The candidate gene tar-1 (for Trichostrongylus acetylcholine receptor subunit gene 1) comprising 13 704 base pairs was thus identified. BLAST comparison of the sequenced clone with GenBank, followed by comparison of translated regions in six reading frames with protein databases, identified clearly defined tracts corresponding to 12 putative exons sharing high sequence homology to other nAChR genes and able to code for sequential regions of a putative nAChR α-subunit protein (tar-1). Tar-1 shares sequence similarities with over 40 nAChR subunit proteins. The highest similarity (91.6%) is with unc-38, suggesting that nAChR sequences from nematodes are closely related. The sequence includes motifs typical of these molecules including adjacent cysteine residues at the ACh binding site and four transmembrane regions. The DNA sequence presents the longest genomic tract described for this organism and should prove useful as a probe source in the search for nAChR genes from this and other nematodes and for studying the molecular mechanism of resistance to levamisole, a drug which is known to act on nAChRs of worms and which is widely used for parasite control.

Determination of the Gene Sequence and the Three-dimensional Structure at 2.4 Å Resolution of Methanol Dehydrogenase from MethylophilusW3A1

The DNA sequences for the genes encoding the heavy and light subunits of methanol dehydrogenase from Methylophilus methylotrophusW3A1 have been determined. The deduced amino acid sequence has enabled the structure of the enzyme to be refined at 2.4 Å resolution against X-ray data collected on a Hamlin area detector. The structure was refined using the programs PROFFT and X-PLOR with several model building step interspersed. The final model contains two heavy chains (571 amino acids), two light chains (69 amino acids), two molecules of pyrroloquinoline quinone, two Ca 2+and 521 solvent molecules. Each half molecule contains four disulfide linkages and four cispeptides. One of the disulfides is formed from two adjacent cysteine residues linked by a trans peptide which creates a novel eight-membered ring. The heavy subunit is an 8-fold β-propeller, each “blade” of which is a four-stranded antiparallel twisted β-sheet. The light chain is an elongated subunit stretching across the surface of the heavy subunit, with residues 1 to 32 containing four β-turns and residues 33 to 62 forming a helix; however, it neither interacts with the active site, nor the other HL dimer and its functional role is obscure. Around the 8-fold β-propeller there is a repeating pattern of tryptophan residues located in the outer strand of seven of the eight β-leaflets, each packed between adjacent leaflets. Each of these tryptophan residues is centered in the β-strand and participates in the main chain hydrogen bonding of the sheet. Five of the seven tryptophan residues have closely similar interactions with the adjacent β-leaflet including stacking of the tryptophan indole rings against a peptide plane and formation of a hydrogen bond from NE1 of the indole ring to a main-chain carbonyl. This repeating pattern is conserved over a number of MEDH sequences. The PQQ is located on the pseudo 8-fold rotation axis of the heavy subunit, in a funnel-shaped internal cavity, sandwiched between the indole ring of Trp237 and the two sulfur atoms of the Cys103-Cys104 vicinal disulfide. A hexacoordinate Ca 2+is bound in the active site by one nitrogen and five oxygen ligands, three from the PQQ and the others from two protein side-chains. In the active site an isolated solvent molecule is bound to the O5 of PQQ and to a nearby aspartate side-chain; its position may be the binding site for methanol. The aspartate might than serve as a general base for proton abstraction from the substrate hydroxyl. The C5 atom of PQQ could be activated by electrophilic catalysis by a nearby argenine side-chain or by the calcium ion bound to PQQ.

The Free Radical of the Anaerobic Ribonucleotide Reductase from Escherichia coli Is at Glycine 681

The anaerobic ribonucleoside triphosphate reductase of Escherichia coli is an iron-sulfur protein carrying an oxygen-sensitive organic radical, which is essential for catalysis. The radical was tentatively proposed to be on glycine 681, based on a comparison with the glycyl radical-containing enzyme pyruvate formate-lyase. By EPR spectroscopy of selectively 2H- and 13C-labeled anaerobic ribonucleotide reductase, the radical was now unambiguously assigned to carbon-2 of a glycine residue. The large 1H hyperfine splitting (1.4 millitesla) was assigned to the alpha-proton. Site-directed mutagenesis was used to change glycine 681 into an alanine residue. In separate experiments, the two adjacent residues, cysteine 680 and tyrosine 682, were changed into serine and phenylalanine, respectively. All mutated proteins were retained on dATP-Sepharose, indicating that the mutant proteins had intact allosteric sites. They also contained amounts of iron comparable with the wild type reductase and showed the same iron-sulfur-related spectrum, suggesting that the mutant proteins were properly folded. Of the three mutant proteins only the G681A protein completely lacked the detectable glycyl radical as well as enzyme activity. Our results identify glycine 681 as the stable free radical site in E. coli anaerobic ribonucleotide reductase.

Racemization of an L-phenylalanine residue catalysed by an adjacent cysteine in a bradykinin peptide antagonist

A number of peptides and proteins contain aromatic amino acid residues adjacent to a cysteine group in their primary sequence. Analysis of the ratios of D- to L-phenylalanine residues in some bradykinin antagonists has revealed that racemization occurs during conventional strong acid hydrolysis of the parent peptides. The mechanism for this unusual transformation appears to involve intramolecular catalysis by an adjacent cysteine residue.

Processing and evolution of the N-terminal region of the arterivirus replicase ORF1a protein: identification of two papainlike cysteine proteases

Two adjacent papainlike cysteine protease (PCP) domains, PCP alpha and PCP beta, were identified in the N-terminal region of the open reading frame 1a replicase proteins of the arteriviruses porcine reproductive and respiratory syndrome virus and lactate dehydrogenase-elevating virus. The replicase of the related virus equine arteritis virus contains only one active PCP in the corresponding region. Sequence comparison revealed that the equine arteritis virus PCP alpha counterpart probably was inactivated by loss of its catalytic Cys residue. For both porcine reproductive and respiratory syndrome virus and lactate dehydrogenase-elevating virus, the generation of two processing products, nsp1 alpha and nsp1 beta, was demonstrated by in vitro translation. Site-directed mutagenesis and sequence comparison were used to identify the putative active-site residues of the PCP alpha and PCP beta protease domains and to show that they mediate the nsp1 alpha/1 beta and nsp1 beta/2 cleavages, respectively.

Effect of adjacent histidine and cysteine residues on the spontaneous degradation of asparaginyl- and aspartyl-containing peptides.

Aspartate and asparagine residues in polypeptides are subject to nonenzymatic reactions that lead to deamidation, isomerization, peptide bond cleavage and racemization. Much of this reactivity is due to the propensity for the initial formation of a cyclic succinimide intermediate. We have been interested in determining the effect of the side chains of neighboring histidine and cysteine residues in facilitating these reactions, particularly in the possibility that they can act as general acids and bases. In this study, we found little or no effect of histidine residues preceding an asparagine residue in hexapeptides derived from the sequence of adrenocorticotropic hormone, while a histidine residue preceding an aspartic acid residue was found to increase the rate of succinimide formation 8- to 11-fold. The presence of a histidine residue following either an asparagine or aspartic acid residue did not effect the rate of succinimide formation by peptide-bond nitrogen attack, but did increase the rate of the competing side-chain nitrogen attack leading to cleavage in the asparaginyl-containing peptide. We found that the effect of a cysteine residue following an asparagine or aspartic acid residue was in general similar to that of a serine residue, although the cleavage reaction appeared to be enhanced. These results suggest that His-Asp sequences may be particularly labile to spontaneous degradation in proteins and peptides, possibly owing to the ability of the histidine residue to facilitate succinimide formation by protonating the OH- leaving group on the side chain carboxylic acid of the aspartic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Mutations of some critical amino acid residues in the hepatitis B virus surface antigen.

Amino acid substitutions at several positions in the surface antigen (HBsAg) of hepatitis B virus (HBV) in natural isolates and the products of recombinant DNA molecules have identified important residues for cross-reaction with specific antibodies (anti-HBs) and the induction of antibodies with certain serological specificities. In a further group of mutants described here, cysteine residues in a region believed to be significant of the a epitope have been changed to serines. Of the three adjacent cysteine residues at positions 137, 138 and 139, mutation of either of the flanking residues reduced cross-reactivity with polyclonal anti-HBs, while alteration of the central residue was relatively well-tolerated. Mutation of cysteine 149 to serine or of glycine 145 to arginine (imitating naturally occurring mutants), lysine, or glutamatic acid all led to loss of cross-reactivity with polyclonal antisera.

The role of the novel disulphide ring in the active site of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens.

All cysteines in methanol dehydrogenase (MDH) from Methylobacterium extorquens are involved in intra-subunit disulphide bridge formation. One of these is between adjacent cysteine residues which form a novel ring structure in the active site. It is readily reduced, the reduced enzyme being inactive in electron transfer to cytochrome cL. The inactivation is not a result of major structural change or to modification of the prosthetic group pyrrolo-quinoline quinone (PQQ). The reduced enzyme appears to remain active with the artificial electron acceptor phenazine ethosulphate but this is because the dye re-oxidizes the adjacent thiols back to the original disulphide bridge. No free thiols were detected during the reaction cycle with cytochrome cL. Carboxymethylation of the thiols produced by reduction of the novel disulphide ring led to formation of active enzyme. Reconstitution of inactive Ca(2+)-free MDH with Ca2+ led to active enzyme containing the oxidized bridge and reduced quinol, PQQH2, consistent with the conclusion that no hydrogen transfer occurs between these groups in the active site. It is concluded that the disulphide ring in the active site of MDH does not function as a redox component of the reaction. The disulphide ring has no special function in the process of Ca2+ incorporation into the active site. It is suggested that this novel structure might function in the stabilization or protection of the free radical semiquinone form of the prosthetic group (PQQH.) from solvent at the entrance to the active site.

Chemokines, a family of chemotactic cytokines.

Chemokines are low-molecular-weight proteins that stimulate recruitment of leukocytes. They are secondary pro-inflammatory mediators that are induced by primary pro-inflammatory mediators such as interleukin-1 (IL-1) or tumor necrosis factor (TNF). The physiologic importance of this family of mediators is derived from their specificity. Unlike the classic leukocyte chemo-attractants, which have little specificity, members of the chemokine family induce recruitment of well-defined leukocyte subsets. Thus, chemokine expression can account for the presence of different types of leukocytes observed in various normal or pathologic states. There are two major chemokine sub-families based upon the position of cysteine residues, i.e., CXC and CC. All members of the CXC chemokine sub-family have an intervening amino acid between the first two cysteines; members of the CC chemokine sub-family have two adjacent cysteines. As a general rule (with some notable exceptions), members of the CXC chemokines are chemotactic for neutrophils, and CC chemokines are chemotactic for monocytes and a small sub-set of lymphocytes. This review discusses the potential role of chemokines in inflammation and focuses on the two best-characterized chemokines, monocyte chemoattractant protein-1 (MCP-1), a CC chemokine, and interleukin-8 (IL-8), a member of the CXC chemokine sub-family.