Control of 5-aminolaevulinate synthetase activity in Rhodopseudomonas spheroides a role for trisulphides.
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1. The aminolaevulinate synthetase activator of fresh extracts of semi-anaerobically grown Rhodopseudomonas spheroids was resolved into two fractions by ion-exchange chromatography. One fraction was identified as cystine trisulphide (CySSSCy). Analysis of the other fraction indicated the presence of about equal amounts of glutathione trisulphide (GSSSG) and the mixed trisulphide of glutathione and cystine (GSSSCy). 2. Four further fractions with activator activity were observed on ion-exchange chromatography of extracts prepared by methods similar to those described earlier [Neuberger et al. (1973)Biochem. J. 136,491-499]. These activators were generated by the extraction procedure. Two of them have been identified as trisulphanedisulphonate (S5O62-) and additional cystine trisulphide. 3. For the series of polysulphanedisulphonates (-O3S-Sn-SO3-, n greater than or equal to 1), activator activity at muM concentrations was exhibited only by compounds with n greater than 3. This, together with the results described above, indicates that for a compound R-Sn-R' (where R and R' are organic or inorganic groups) the only structural requirement for activity is n greater than or equal to 3. 4. Oxygenation of a semipanaerobic culture of R. spheroids for 1.5h before harvesting the cells produced a decrease of more than 90% in the cellular content of cystine trisulphide and glutathione trisulphides. 5. Chromatography on DEAE-Sephadex confirmed the presence of multiple forms of aminolaevulinate synthetase in extracts of R. spheroides [Tuboi et al. (1970) Arch.Biochem. Biophys. 138,147-154]. Oxygenation of a semi-anaerobic culture resulted in the disappearance of high-activity enzyme (a-form) and the accumulation of low-activity enzyme (b-form) in the cell. Spontaneous activation [Marriott et al. (1969) Biochem. J. 111,385-394] And activation by cystine trisulphide both resulted in the almost complete conversion of the b-form into the a-form. (+info)
The major transition state in folding need not involve the immobilization of side chains.
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During protein folding in which few, if any, definable kinetic intermediates are observable, the nature of the transition state is central to understanding the course of the reaction. Current experimental data does not distinguish the relative contributions of side chain immobilization and dehydration phenomena to the major rate-limiting transition state whereas this distinction is central to theoretical models that attempt to simulate the behavior of proteins during folding. Renaturation of the small proteinase inhibitor cystatin under oxidizing versus reducing conditions is the first experimental case in which these processes can be studied independently. Using this example, we show that sidechain immobilization occurs downstream of the major folding transition state. A consequence of this is the existence of states with disordered side chains, which are distinct from kinetic protein folding intermediates and which lie within the folded state free energy well. (+info)
Role of ligand substitution on long-range electron transfer in azurins.
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Azurin contains two potential redox sites, a copper centre and, at the opposite end of the molecule, a cystine disulfide (RSSR). Intramolecular electron transfer between a pulse radiolytically produced RSSR- radical anion and the blue Cu(II) ion was studied in a series of azurins in which single-site mutations were introduced into the copper ligand sphere. In the Met121His mutant, the rate constant for intramolecular electron transfer is half that of the corresponding wild-type azurin. In the His46Gly and His117Gly mutants, a water molecule is co-ordinated to the copper ion when no external ligands are added. Both these mutants also exhibit slower intramolecular electron transfer than the corresponding wild-type azurin. However, for the His117Gly mutant in the presence of excess imidazole, an azurin-imidazole complex is formed and the intramolecular electron-transfer rate increases considerably, becoming threefold faster than that observed in the native protein. Activation parameters for all these electron-transfer processes were determined and combined with data from earlier studies on intramolecular electron transfer in wild-type and single-site-mutated azurins. A linear relationship between activation enthalpy and activation entropy was observed. These results are discussed in terms of reorganization energies, driving force and possible electron-transfer pathways. (+info)
Membranolytic selectivity of cystine-stabilized cyclic protegrins.
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To correlate conformational rigidity with membranolytic selectivity of antimicrobial activity and cytotoxicity, we prepared six cyclic analogs of protegrin-1 (PG-1), an 18-residue cationic peptide with a broad-spectrum antimicrobial activity. These cyclic protegrins bear end-to-end peptide bonds together with varying numbers (zero to three) of cross-strand disulfide constraints. The most constrained analog is a cyclic tricystine protegrin (ccPG 3) containing three evenly spaced, parallel disulfide bonds. Antimicrobial assays against 10 organisms in low- and high-salt conditions showed that these cyclic protegrins were broadly active with different antimicrobial profiles against Gram-positive and Gram-negative bacteria, fungi and one tested virus, HIV-1. Compared to PG-1, the cyclic tricystine ccPG 3 displayed approximately a 10-fold decrease in hemolytic activity against human cells and 6- to 30-fold improvement of membranolytic selectivity against six of the 10 tested organisms. In contrast, [DeltaSS]cPG 8, a cyclic protegrin with no disulfide bond, and [DeltaCys6,15]cPG 5, a cyclic mimic of PG-1 with one disulfide bond, exhibited activity spectra, potency, and cytotoxicity similar to PG-1. Circular dichroism showed that cyclic protegrins containing with one to three cystine bonds displayed some degree of beta-strand structures in water/trifluoroethanol or phosphate-buffered solutions. Collectively, our results indicate that cyclic structures are useful in the design of antimicrobial peptides and that an increase in the conformational rigidity of protegrins may confer membranolytic selectivity that dissociates antimicrobial activity from hemolytic activity. (+info)
Localization of disulfide bonds in the cystine knot domain of human von Willebrand factor.
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von Willebrand factor (VWF) is a multimeric glycoprotein that is required for normal hemostasis. After translocation into the endoplasmic reticulum, proVWF subunits dimerize through disulfide bonds between their C-terminal cystine knot-like (CK) domains. CK domains are characterized by six conserved cysteines. Disulfide bonds between cysteines 2 and 5 and between cysteines 3 and 6 define a ring that is penetrated by a disulfide bond between cysteines 1 and 4. Dimerization often is mediated by additional cysteines that differ among CK domain subfamilies. When expressed in a baculovirus system, recombinant VWF CK domains (residues 1957-2050) were secreted as dimers that were converted to monomers by selective reduction and alkylation of three unconserved cysteine residues: Cys(2008), Cys(2010), and Cys(2048). By partial reduction and alkylation, chemical and proteolytic digestion, mass spectrometry, and amino acid sequencing, the remaining intrachain disulfide bonds were characterized: Cys(1961)-Cys(2011) (), Cys(1987)-Cys(2041) (), Cys(1991)-Cys(2043) (), and Cys(1976)-Cys(2025). The mutation C2008A or C2010A prevented dimerization, whereas the mutation C2048A did not. Symmetry considerations and molecular modeling based on the structure of transforming growth factor-beta suggest that one or three of residues Cys(2008), Cys(2010), and Cys(2048) in each subunit mediate the covalent dimerization of proVWF. (+info)
The effect of dietary sulfur-containing amino acids on the activity of drug-metabolizing enzymes in rat-liver microsomes.
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Male Wistar rats were fed either a balanced diet whose protein source was a mixture of amino acids (diet 1), or a similar diet which differed only in having 48% less the quantity of the sulfur-containing amino acids methionine and cystine (diet 2). The diets were given either continuously for 1 month or for 15 days after a protein-free diet. Both diets 1 and 2 permitted good growth of rats and relatively stable microsomal protein content. Protein depletion decreased the total proteins, total phospholipids, and cytochrome P-450 content, and it strongly increased UDP-glucuronosyltransferase activity in rat-liver microsomes. Repletion with diet 1 restored those values, to the level found in control rats. However, diet 2, given continuously induced an increase in UDP-glucuronosyltransferase activity and in the cytochrome P-450 concentration. Since high UDP-glucuronosyltransferase activity was related to lessened amounts of sulfur-containing amino acids in the diet, we discuss the possible effect of methionine and cystine on the regulation of glucuronoconjugation in relation to sulfoconjugation. (+info)
Purification, structure determination and synthesis of covalitoxin-II, a short insect-specific neurotoxic peptide from the venom of the Coremiocnemis validus (Singapore tarantula).
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Spider venoms contain toxins that specifically immobilize and kill insects. We report the purification and characterization of a new insect-specific toxin named covalitoxin-II (Cvtx-II; mass, 3406. 24+/-0.64), from Coremiocnemis validus (Singapore tarantula) venom. The complete 31 amino acid sequence of Cvtx-II has been determined and it shows less than 40% identity with spider toxins. However, Cvtx-II has conserved cystine motif analogous to other spider and omega-conotoxins. Cvtx-II was chemically synthesized and identified with the native Cvtx-II. Synthetic Cvtx-II induced insect-specific non-lethal excitatory activity when injected into crickets, but not in cockroaches and mice. (+info)
Predicting the oxidation state of cysteines by multiple sequence alignment.
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MOTIVATION: Protein sequences found in databanks usually do not report post translational covalent modifications such as the oxidation state of cystein (Cys) residues. Accurate prediction of whether a functionally or structurally important Cys occurs in the oxidized or thiol form would be helpful for molecular biology experiments and structure prediction. RESULTS: A new method is presented for predicting the oxidation state of Cys residues based on multiple sequence alignments and on the observation that Cys tends to occur in the same oxidation state within the same protein. The prediction of the redox state of Cys performs above 82%. The oxidation state of Cys correlates with the cellular location of the given protein within the cell, but the correlation is not perfect (up to 70%). We also perform a statistical analysis of the different redox states of Cys found in secondary structures and buried positions, and of the secondary structures linked by disulfide bonds. The results suggest that the natural borderline lies between the different oxidation states of Cys rather than between the half cystines and cysteins. AVAILABILITY: A web server implementing the prediction method is available at http://guitar.rockefeller.edu/approximately andras/cyspred.html CONTACT: [email protected] (+info)