Additional Disulfide Bond Research Articles

Thermostable variants of bovine beta-lactoglobulin.

The thermal stability of bovine beta-lactoglobulin (BLG) has been enhanced by the introduction of an additional disulfide bond. Wild-type BLG has two disulfide bonds, C106-C119 and C66-C160, with a free cysteine at position 121. We have designed, with the aid of molecular modeling calculations, two mutants of a recombinant BLG (rBLG), L104C and A132C. Molecular dynamics simulations were performed at 300K to study the effect of these alterations on the conformation of the protein. These mutants were then created by site-directed mutagenesis and purified from Escherichia coli carrying a tac expression vector using a two-step renaturation method. Formation of disulfide linkages in the correct arrangement, as designed, was confirmed by peptide mapping. In contrast to wild-type rBLG, which polymerizes at temperatures > 65 degrees C, neither of the mutant proteins polymerized. The conformational stability of the L104C and A132C mutant proteins against thermal denaturation has been substantially increased (8-10 degrees C) as compared with wild-type rBLG. Furthermore, the A132C rBLG exhibits an enhanced stability against denaturation by guanidine hydrochloride as compared with the wild-type or L104C rBLG.

A genetically engineered human IgG with limited flexibility fully initiates cytolysis via complement

A causal link between antigen induced conformational change and functional activity has been previously suggested for the triggering of immunoglobulin effector functions. To test this hypothesis an immunoglobulin with greatly restricted Fab flexibility has been created by site-directed mutagenesis. Serine 119, a residue at the elbow in the first heavy chain constant domain of a mouse-human chimeric immunoglobulin, was replaced by cysteine 119. The resulting mutant immunoglobulin had an additional intramolecular disulfide bond connecting the two heavy chains. This newly introduced covalent bond between antigen binding arms gave rise to a compact ‘tethered’ conformation which displayed lowered segmental flexibility as determined by nanosecond fluorescence depolarization anisotropy. However, the ability of this tethered immunoglobulin to initiate lysis of target cells via the complement cascade was unimpaired. Therefore, it is likely that allosteric or distortive mechanisms of conformational change induced complement activation which require unhindered ‘twist’ and/or ‘waggle’ motions of Fab are untenable.

The Role of Amino-Terminal Disulfide Bonds in the Structure and Assembly of Human Fibrinogen

Human fibrinogen contains two half-molecules, each composed of an α, β, and γ chain linked by disulfide bonds. The two half-molecules (αβγ) are held together in the native protein by additional disulfide bonds located in the amino terminus of each chain. Site-directed mutagenesis, in which the amino-terminal Cys residues (α-Cys28 and 36, β-Cys65, and γ-Cys8 and 9) were converted to Ser, was carried out in order to study the role of the amino-terminal disulfide bonds in the structure and assembly of fibrinogen. An analysis of the fibrinogen synthesized in transfected baby hamster kidney (BHK) cells employing various combinations of these mutations revealed that α-Cys36 and β-Cys65 form disulfide bonds between two αβγ half-molecules, rather than within the same half-molecule; furthermore, these two disulfide bonds are sufficient to hold the two αβγ half-molecules together as intact fibrinogen. Disulfide bonds formed by γ-Cys8 and 9 were also sufficient to hold the two fibrinogen αβγ half-molecules together, while the disulfide bond between the two α-Cys28 residues failed to form in the absence of the disulfide bonds linking the α and β chains and the two γ chains.

X-ray crystal structure of canine myeloperoxidase at 3 Å resolution

The three-dimensional structure of the enzyme myeloperoxidase has been determined by X-ray crystallography to 3 Å resolution. Two heavy atom derivatives were used to phase an initial multiple isomorphous replacement map that was subsequently improved by solvent flattening and non-crystallographic symmetry averaging. Crystallographic refinement gave a final model with an R-factor of 0.257. The root-mean-square deviations from ideality for bond lengths and angles were 0.011 Å and 3.8 °. Two, apparently identical, halves of the molecule are related by local dyad and covalently linked by a single disulfide bridge. Each half-molecule consists of two polypeptide chains of 108 and 466 amino acid residues, a heme prosthetic group, a bound calcium ion and at least three sites of asparagine-linked glycosylation. There are six additional intra-chain disulfide bonds, five in the large polypeptide and one in the small. A central core region that includes the heme binding site is composed of five α-helices. Regions of the larger polypeptide surrounding this core are organized into locally folded domains in which the secondary structure is predominantly α-helical with very little organized β-sheet. A proximal ligand to the heme iron atom has been identified as histidine 336, which is in turn hydrogen-bonded to asparagine 421. On the distal side of the heme, histidine 95 and arginine 239 are likely to participate directly in the catalytic mechanism, in a manner analogous to the distal histidine and arginine of the non-homologous enzyme cytochrome c peroxidase. The site of the covalent linkage to the heme has been tentatively identified as glutamate 242, although the chemical nature of the link remains uncertain. The calcium binding site has been located in a loop comprising residues 168 to 174 together with aspartate 96. Myeloperoxidase is a member of a family of homologous mammalian peroxidases that includes thyroid peroxidase, eosinophil peroxidase and lactoperoxidase. The heme environment, defined by our model for myeloperoxidase, appears to be highly conserved in these four mammalian peroxidases. Furthermore, the conservation of all 12 cysteine residues involved in the six intra-chain disulfide bonds and the calcium binding loop suggests that the three-dimensional structures of members of this gene family are likely to be quite similar.

Labile disulfide bonds in human placental insulin receptor.

The disulfide crosslinking pattern of human placental insulin receptor was investigated using selective reduction with tributylphosphine followed by alkylation with N-[3H]ethylmaleimide. Insulin receptor contains a single sulfhydryl group in each beta subunit whose alkylation with N-[3H]ethylmaleimide inhibits receptor autophosphorylation. Alkylation is partially inhibited by ATP or the nonhydrolyzable substrate analog adenosine 5'-[beta,gamma-imido]triphosphate when the nucleotides are added as Mn2+ complexes. Neither insulin nor 6 M guanidinium chloride renders additional sulfhydryl groups accessible to alkylation. When the receptor is reduced under drastic conditions with tributylphosphine in guanidinium chloride, 32 of the 37 sulfhydryl groups in the receptor's alpha subunit can be alkylated with N-[3H]ethylmaleimide. Surprisingly only three of the 10 cysteines in the beta subunit become titratable under identical conditions. By using highly selective reducing conditions, we were able to determine quantitatively the maximum number of disulfide bridges that link the two alpha beta halves to form the tetrameric structure and those that couple the alpha to the beta subunits. Liberation of two sulfhydryl groups in the alpha and one in the beta subunit resulted in formation of alpha beta dimers. Free beta subunit was formed when an additional disulfide bond was reduced. It is remarkable that the tetrameric structure of this highly complex receptor molecule, which contains a large number of cysteine residues, is maintained by such a small number of disulfide bonds. Three models of the arrangement of the labile disulfide bonds, consistent with these findings, are proposed.

Disulfide Reduction and Molecular Dissociation Improves the Proteolysis of Soy Glycinin by Pancreatin in vitro

ABSTRACTTHE EFFECTS of structural modification by urea and dithiothreitol on the in vitro pancreatin proteolysis of soy glycinin was studied. Urea, up to 4.5M, which causes dissociation of glycinin into subunits and some unfolding of the polypeptides progressively increased proteolysis by pancreatin as measured by the pH‐stat method. Reduction of the intermolecular disulfide bonds with dithiothreitol doubled the rate of proteolysis and when additional intramolecular disulfide bonds of glycinin were cleaved, the rate of digestibility increased approximately threefold becoming equivalent to casein in its susceptibility to proteolysis by pancreatin.

Primary Structure of Bovine Carboxypeptidase B: INFERENCES FROM THE LOCATIONS OF THE HALF-CYSTINES AND IDENTIFICATION OF THE ACTIVE SITE ARGININE

Abstract Bovine pancreatic carboxypeptidase B is a protein which is believed to have evolved with carboxypeptidase A by gene duplication from a common ancestral prototype enzyme. Whereas bovine carboxypeptidase A has but one disulfide bond, carboxypeptidase B has three. Reduced, S-aminoethylated carboxypeptidase B was maleylated and the product subjected to tryptic hydrolysis. The peptides with COOH-terminal arginine so produced were fractionated by gel filtration and by chromatography on DEAE-Sephadex A-25. Eleven of the 14 principal peptides possible were isolated. The peptides that contained the six S-aminoethylcysteine residues originating from the half-cystines in the enzyme were subjected to sequence analysis and were aligned on the basis of substantial homologies with sequences in carboxypeptidase A. Referred to the sequence of carboxypeptidase A, the half-cystines in carboxypeptidase B are at positions 66, 79, 138, 152, 161, and 166. Those at positions 138 and 161 are homologous with the two half-cystine residues in carboxypeptidase A. The remaining four half-cystines, when related to the known chain conformation of carboxypeptidase A, are in locations which would be stereochemically compatible with the presence of two additional disulfide bonds in the structure: one, as previously found, between half-cystines 66 and 79 and the other between half-cystines 152 and 166. One of the half-cystines is present in a peptide which terminates in an arginine residue recognized to be homologous with Arg-145 in carboxypeptidase A, a residue critical for the establishment of the specificity of carboxypeptidase A. The present experiments have permitted placement in sequence of an additional 85 residues in carboxypeptidase B, bringing the total residues so placed to 221. Of these residues, 100 are homologous with residues in carboxypeptidase A. These homologies, the configurational correlations revealed by the positions of the half-cystines, the conservation of at least two of the three residues involved in zinc binding (His-69 and Glu-72) and of the three functionally essential residues in the active site (Arg-145, Tyr-248 and Glu-270) all implicate the existence of a close structural homology with carboxypeptidase A.