995 resultados para chloride binding
Resumo:
The conformational stability of Plasmodium falciparum triosephosphate isomerase (TIMWT) enzyme has been investigated in urea and guanidinium chloride (GdmCl) solutions using circular dichroism, fluorescence, and size-exclusion chromatography. The dimeric enzyme is remarkably stable in urea solutions. It retains considerable secondary, tertiary, and quaternary structure even in 8 M urea. In contrast, the unfolding transition is complete by 2.4 M GdmCl. Although the secondary as well as the tertiary interactions melt before the perturbation of the quaternary structure, these studies imply that the dissociation of the dimer into monomers ultimately leads to the collapse of the structure, suggesting that the interfacial interactions play a major role in determining multimeric protein stability. The Cm(urea)/Cm(GdmCl) ratio (where Cm is the concentration of the denaturant required at the transition midpoint) is unusually high for triosephosphate isomerase as compared to other monomeric and dimeric proteins. A disulfide cross-linked mutant protein (Y74C) engineered to form two disulfide cross-links across the interface (13-74‘) and (13‘-74) is dramatically destablized in urea. The unfolding transition is complete by 6 M urea and involves a novel mechanism of dimer dissociation through intramolecular thiol−disulfide exchange.
Resumo:
The binding of 1-anilino-8-naphthalene-sulfonic acid to globular proteins at acidic pH has been investigated by electrospray ionization mass spectrometry (ESIMS). Mass spectra of apomyoglobin recorded in the pH range 2−7 establish that maximal ANS binding is observed at pH 4.0. As many as seven distinct species may be observed in the gas phase which correspond to protein molecules containing one to six molecules of bound ANS. At neutral pH only a single molecule of ANS is bound. In the case of cytochrome c, maximal binding is observed at pH 4.0, with five molecules being bound. Binding is suppressed at neutral pH. In both cases ESIMS demonstrates maximal ANS binding at pH values where the proteins have been reported to exist in molten globule states. ANS binding is not observed for lysozyme, which has a tightly folded structure over the entire pH range. Reduction of disulfide bonds in lysozyme leads to the detection of ANS-bound species at neutral pH. Binding is suppressed at low pH due to complete unfolding of the reduced protein. The results suggest that ESIMS may provide a convenient method of probing the stoichiometry and distribution of dye complexes with molten protein globules
Resumo:
A phosphorylcholine-binding protein from the hemolymph of the snail Achatina fulica was purified to near homogeneity using a Sepharose phenylphosphorylcholine affinity column. The protein bound to the affinity column was eluted with 5 mM phosphorylcholine as a single symmetrical peak. The purified protein (400 Kda) contained 35–40% carbohydrate. On SDS-PAGE the protein separated into two bands of 20 and 24 Kda, and had a pI of 5.9. On immunodiffusion, antiserum to the snail phosphorylcholine binding protein did not cross-react against other phosphorylcholine binding proteins, like rat serum phosphorylcholine-binding protein (PCBP), limulus C-reactive protein (CRP), or human CRP. On pretreatment of the snail hemolymph with this antiserum, the hemagglutination titer of the hemolymph was markedly decreased. The purified snail phosphorylcholine binding protein agglutinated rabbit erythrocytes in the absence of divalent cation (Ca+2) but trace amount of Ca+2 increased its binding. The strongest inhibitor of the agglutination reaction was lactose, followed by melibiose and 2-deoxygalactose. The relationships of the snail phosphorylcholine binding protein to other hemolymph agglutinins and to CRPs are discussed in light of common phylogeny.
Resumo:
Computer-modelling studies on the modes of binding of the three guanosine monophosphate inhibitors 2'-GMP, 3'-GMP, and 5'-GMP to ribonuclease (RNase) T1 have been carried out by energy minimization in Cartesian-coordinate space. The inhibitory power was found to decrease in the order 2'-GMP > 3'-GMP > 5'-GMP in agreement with the experimental observations. The ribose moiety was found to form hydrogen bonds with the protein in all the enzyme-inhibitor complexes, indicating that it contributes to the binding energy and does not merely act as a spacer between the base and the phosphate moieties as suggested earlier. 2'-GMP and 5'-GMP bind to RNase T1 in either of the two ribose puckered forms (with C3'-endo more favoured over the C2'-endo) and 3'-GMP binds to RNase T1 predominantly in C3'-endo form. The catalytically important residue His-92 was found to form hydrogen bond with the phosphate moiety in all the enzyme-inhibitor complexes, indicating that this residue may serve as a general acid group during catalysis. Such an interaction was not found in either X-ray or two-dimensional NMR studies.
Resumo:
SHMT (serine hydoxymethyltransferase), a type I pyridoxal 5'-phosphate-dependent enzyme, catalyses the conversion of L-serine and THF (tetrahydrofolate) into glycine and 5,10 -methylene THE SHMT also catalyses several THF-independent side reactions such as cleavage of P-hydroxy amino acids, trans-amination, racemization and decarboxylation. In the present study, the residues Asn(341), Tyr(60) and Phe(351), which are likely to influence THF binding, were mutated to alanine, alanine and glycine respectively, to elucidate the role of these residues in THF-dependent and -independent reactions catalysed by SHMT. The N341A and Y60A bsSHMT (Bacillus stearothermophilus SHMT) mutants were inactive for the THF-dependent activity, while the mutations had no effect on THF-independent activity. However, mutation of Phe(351) to glycine did not have any effect oil either of the activities. The crystal structures of the glycine binary complexes of the mutants showed that N341A bsSHMT forms an external aldimine as in bsSHMT, whereas Y60A and F351G bsSHMTs exist as a Mixture of internal/external aldimine and gem-diamine forms. Crystal structures of all of the three Mutants obtained in the presence of L-allo-threonine were similar to the respective glycine binary complexes. The structure of the ternary complex of F351G bsSHMT with glycine and FTHF (5-formyl THF) showed that the monoglutamate side chain of FTHF is ordered in both the subunits of the asymmetric unit, unlike in the wild-type bsSHMT. The present studies demonstrate that the residues Asn(341) and Tyr(60) are pivotal for the binding of THF/FTHF, whereas Phe(351) is responsible for the asymmetric binding of FTHF in the two subunits of the dimer.
Resumo:
Salmonella typhimurium YeaD (stYeaD), annotated as a putative aldose 1-epimerase, has a very low sequence identity to other well characterized mutarotases. Sequence analysis suggested that the catalytic residues and a few of the substrate-binding residues of galactose mutarotases (GalMs) are conserved in stYeaD. Determination of the crystal structure of stYeaD in an orthorhombic form at 1.9 angstrom resolution and in a monoclinic form at 2.5 angstrom resolution revealed this protein to adopt the beta-sandwich fold similar to GalMs. Structural comparison of stYeaD with GalMs has permitted the identification of residues involved in catalysis and substrate binding. In spite of the similar fold and conservation of catalytic residues, minor but significant differences were observed in the substrate- binding pocket. These analyses pointed out the possible role of Arg74 and Arg99, found only in YeaD-like proteins, in ligand anchoring and suggested that the specificity of stYeaD may be distinct from those of GalMs
Resumo:
A simple and efficient procedure for the purification of the riboflavin-binding protein from hen's egg yolk is described. This method involves the removal by exclusion of lipoproteins and subsequent fractionation of soluble yolk proteins held on a DEAE-cellulose column by a salt gradient which is followed by purification by gel filtration on Sephadex G-100. The protein thus isolated is homogeneous by various physicoehemical, immunological, and functional criteria.
Resumo:
The temperature dependence of the chlorine-35 n.q.r. in the mercuric chloride-4-picoline N-oxide complex has been studied from 77 K to room temperature, and the results are used to assign the observed frequencies to terminal and bridging chlorines.
Resumo:
A pure sample of nitrosyl chloride has been prepared either by reaction of phosphorus trichloride with concentrated nitric acid or by reaction of phosphorus trichloride with sodium nitrate in presence of water. The nitrosyl chloride gas has been characterized by i.r. spectral data and elemental analysis.
Resumo:
Kinetics of the interaction of Au(III) with native calf thymus DNA has been studied spectrophotometrically to determine the kinetic parameters and to examine their dependency on the concentrations of DNA and Au(III), temperature, ionic strength and pH. The reaction is of the first order with respect to both the nucleotide unit of DNA and Au(III) in the stoichiometry of 2∶1 respectively. The rate constants vary with the initial ratio of DNA to Au(III) and is attributed to the effect of free chloride ions and the existence of a number of reaction sites with slight difference in the rate constants. The activation energies of this interaction have been found to be 14–16 kcal/mol. From the effect of ionic strength the reaction is found to occur between a positive and a negative ion in the rate-limiting step. The logarithm of rate constants are the linear function of pH and the slopes are dependent on ther-values. A plausible mechanism has been proposed which involves a primary dissociation of the major existing species (AuCl2(OH)2)−, to give (AuCl2)+ which then reacts with a site in the nucleotide unit of DNA in the rate-liminting step followed by a rapid binding to another site on the complementary strand of the DNA double helix. There exist a number of binding sites with slight difference in reactivity.
Resumo:
A thiamin-binding protein was isolated and characterized from chicken egg white by affinity chromatography on thiamin pyrophosphate coupled to aminoethyl-Sepharose. The high specificity of interaction between the thiamin-binding protein and the riboflavin-binding protein of the egg white, with a protein/protein molar ratio of 1.0, led to the development of an alternative procedure that used the riboflavin-binding protein immobilized on CNBr-activated Sepharose as the affinity matrix. The thiamin-binding protein thus isolated was homogeneous by the criteria of polyacrylamide-gel disc electrophoresis, double immunodiffusion and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, had a mol.wt. of 38,000 +/- 2000 and was not a glycoprotein. The protein bound [14C]thiamin was a molar ratio of 1.0, with dissociation constant (Kd) 0.3 micrometer.