Analyzing glycerol-mediated protein oligomerization by electrospray ionization mass spectrometry

Glycerol is widely used as protein stabilizer, in both local and commercial preparations, so it has become necessary to develop methods for mass spectrometric analysis of protein preparations in the presence of glycerol. However, this stabilizing agent may cause signal suppression when present in high concentrations, and is also known to induce protein supercharging even at low concentrations. This work reports the,use of electrospray ionization (ESI) mass spectrometry to characterize glycerol-mediated protein oligomerization. this phenomenon seems to involve the formation of strong non-covalent interactions between protein and glycerol involving close contact between the monomers, leading to formation of protein oligomers adducted with glycerol molecules under the characteristic analytical conditions of the ESI interface. At high orders of oligomerization a lower number of glycerol molecules is required to maintain the high oligomeric states than for the dimers and trimers, and it is possible that for the higher oligomers the monomers become so close to one another that non-covalent bonds between the side chains of the amino acid residues in the proteins may be established. Copyright (C) 2005 John Wiley & Sons, Ltd.

Molecular adaptability of nucleoside diphosphate kinase b from trypanosomatid parasites: stability, oligomerization and structural determinants of nucleotide binding

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Highly selective nickel ethylene oligomerization catalysts based on sterically hindered tris(pyrazolyl)borate ligands

The reaction of TlTp' (Tp' = HB(3-mesitylpyrazolyl)(3)(-) (Tp(Ms)), HB(3-mesitylpyrazolyl)(2)(5-mesitylpyrazolyl)(-) (Tp(Ms)*)) with NiCl(2).6H(2)O affords Tp(Ms)NiCl (1) and Tp(Ms)*NiCl (2) in good yield. The compound 2 undergoes an isomerization process to form [{Tp(Ms)**}NiCl](2) (3) (Tp(Ms)** = HB(5-mesitylpyrazolyl)(2)(3-mesitylpyrazolyl)(-)) in 68% yield. Treatment of the tris(pyrazolyl)-borate nickel compounds 1 and 2 with alkylaluminum cocatalysts such as methylalumoxane (MAO) and trimethylaluminum (TMA) in toluene generates active catalysts for ethylene oligomerization. The compound 1 shows turnover frequencies in the range of (2.2-43.1) x 10(3) h(-1). Oligomerization reaction conditions can be adjusted that lead to selectivities as high as 81% for butene-1.

Purification of a PHA-Like chitin-binding protein from Acacia farnesiana seeds: A time-dependent oligomerization protein

A lectin-like protein from the seeds of Acacia farnesiana was isolated from the albumin fraction, characterized, and sequenced by tandem mass spectrometry. The albumin fraction was extracted with 0.5 M NaCl, and the lectin-like protein of A. farnesiana (AFAL) was purified by ion-exchange chromatography (Mono-Q) followed by chromatofocusing. AFAL agglutinated rabbit erythrocytes and did not agglutinate human ABO erythrocytes either native or treated with proteolytic enzymes. In sodium dodecyl sulfate gel electrophoresis under reducing and nonreducing conditions, AFAL separated into two bands with a subunit molecular mass of 35 and 50 kDa. The homogeneity of purified protein was confirmed by chromatofocusing with a pI=4.0+/-0.5. Molecular exclusion chromatography confirmed time-dependent oligomerization in AFAL, in accordance with mass spectrometry analysis, which confers an alteration in AFAL affinity for chitin. The protein sequence was obtained by a liquid chromatography quadrupole time-of-flight experiment and showed that AFAL has 68% and 63% sequence similarity with lectins of Phaseolus vulgaris and Dolichos biflorus, respectively.

Influence of Quaternary Conformation on the Biological Activities of the Asp49-phospholipases A(2)s from Snake Venoms

One of the main components of snake venoms are the Asp49-phospholipases A(2), also known as svPLA(2)s. The study of these toxins is a matter of great scientific interest due to their wide variety of biological effects. In this work we present strong evidences found in literature and other aspects which strengthen the importance of quaternary assembly for understanding the activities and molecular evolution of svPLA(2)s.

Insights into the role of oligomeric state on the biological activities of crotoxin: crystal structure of a tetrameric phospholipase A(2) formed by two isoforms of crotoxin B from Cratalus durissus terrificus venom

Crotoxin B (CB or Cdt PLA(2)) is a basic Asp49-PLA(2) found in the venom of Crotalus durissus terrificus and it is one of the subunits that constitute the crotoxin (Cro). This heterodimeric toxin, main component of the C. d. terrificus venom, is completed by an acidic, nontoxic, and nonenzymatic component (crotoxin A, CA or crotapotin), and it is related to important envenomation effects such as neurological disorders, myotoxicity, and renal failure. Although Cro has been crystallized since 1938, no crystal structure of this toxin or its subunits is currently available. In this work, the authors present the crystal structure of novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2). The results suggest that these assemblies are stable in solution and show that Ser1 and Glu92 of CB1 and CB2, respectively, play an important role in the oligomerization. The tetrameric and dimeric conformations resulting from the association of the isoforms may increase the neurotoxicity of the toxin CB by the creation of new binding sites, which could improve the affinity of the molecular complexes to the presynaptic membrane.

Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structure of chorismate synthase from Mycobacterium tuberculosis

In bacteria, fungi, plants, and apicomplexan parasites, the aromatics compounds, such as aromatics amino acids, are synthesized through seven enzymes from the shikimate pathway, which are absent in mammals. The absence of this pathway in mammals make them potential targets for development of new therapy against infectious diseases, such as tuberculosis, which is the world's second commonest cause of death from infectious disease. The last enzyme of shikimate pathway is the chorismate synthase (CS), which is responsible for conversion of the 5-enolpyruvylshikimate-3-phosphate to chorismate. Here, we report the crystallographic structure of CS from Mycobacterium tuberculosis (MtCS) at 2.65 angstrom resolution. The MtCS structure is similar to other CS structures, presenting beta-alpha-beta sandwich structural topology, in which each monomer of MtCS consists of a central helical core. The MtCS can be described as a tetramer formed by a dimer of dimers. However, analytical ultracentrifugation studies suggest the MtCS is a dimer with a more asymmetric shape than observed on the crystallographic dimer and the existence of a low equilibrium between dimer and tetramer. Our results suggest that the MtCS oligomerization is concentration dependent and some conformational changes must be involved on that event. (c) 2005 Elsevier B.V. All rights reserved.

Profiling the proteome complement of the secretion from hypopharyngeal gland of Africanized nurse-honeybees (Apis mellifera L.)

The protein complement of the secretion from hypopharyngeal gland of nurse-bees (Apis mellifera L.) was partially identified by using a combination of 2D-PAGE, peptide sequencing by MALDI-PSD/MS and a protein engine identification tool applied to the honeybee genome. The proteins identified were compared to those proteins already identified in the proteome complement of the royal jelly of the honey bees. The 2D gel electrophoresis demonstrated this protein complement is constituted of 61 different polypepides, from which 34 were identified as follows: 27 proteins belonged to MRJPs family, 5 proteins were related to the metabolism of carbohydrates and to the oxido-reduction metabolism of energetic Substrates, I protein was related to the accumulation of iron in honeybee bodies and I protein may be a regulator of MRJP-1 oligomerization. The proteins directly involved with the carbohydrates and energetic metabolisms were: alpha glucosidase, glucose oxidase and alpha amylase, whose are members of the same family of enzymes, catalyzing the hydrolysis of the glucosidic linkages of starch; alcohol dehydrogenase and aldehyde dehydrogenase, whose are constituents of the energetic metabolism. The results of the present manuscript support the hypothesis that the most of these proteins are produced in the hypoharyngeal gland of nurse-bees and secreted into the RJ. (C) 2004 Elsevier Ltd. All rights reserved.

Tertiary structural changes of the alpha-hemolysin from Staphylococcus aureus on association with liposome membranes

The interaction of alpha-hemolysin (also called alpha-toxin) from Staphylococcus aureus with mixed egg-yolk phosphatidylcholine/cholesterol liposomes has been investigated using the intrinsic tryptophan fluorescence emission (ITFE) signal. The ITFE intensity of alpha-hemolysin, which was obtained using a novel purification protocol, showed a triphasic increase on incubation with liposomes at low protein/lipid ratios. The first, rapid phase results in an increase in ITFE of 10%, which reflects rapid conformation changes in the alpha-hemolysin on association with the liposome membrane, the second phase of the ITFE increase is associated with a red shift from 334 to 339 nm in the maximum emission wavelength, suggesting the transition to a partially unfolded intermediate in the oligomerization process. The third phase of the ITFE intensity change demonstrates a temporal correlation with the appearance of SDS-stable oligomers. The results demonstrate the feasibility of identification of intermediate protein conformations in complex membrane-associated processes by manipulation of the liposomal membrane composition. (C) 1998 Academic Press.

Crystal structure of a dimeric Ser49 PLA(2)-like myotoxic component of the Vipera ammodytes meridionalis venomics reveals determinants of myotoxicity and membrane damaging activity

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Structural basis for branching-enzyme activity of glycoside hydrolase family 57: Structure and stability studies of a novel branching enzyme from the hyperthermophilic archaeon Thermococcus Kodakaraensis KOD1

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Crystal Structures of Xanthomonas Small Heat Shock Protein Provide a Structural Basis for an Active Molecular Chaperone Oligomer

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Reaction pathway to the synthesis of anatase via the chemical modification of titanium isopropoxide with acetic acid

Anatase nanoparticles were obtained through a modified sol-gel route from titanium isopropoxide modified with acetic acid in order to control hydrolysis and condensation reactions. The modification of Ti(O(i)Pr)(4) with acetic acid reduces the availability of groups that hydrolyze and condense easily through the formation of a stable complex whose structure was determined to be Ti(OCOCH(3))(O(i)Pr)(2) by means of FTIR and (13)C NMR. The presence of this complex was confirmed with FTIR in the early stages of the process. A doublet in 1542 and 1440 cm(-1) stands for the asymmetric and symmetric stretching vibrations of the carboxylic group coordinated to Ti as a bidentate ligand. The gap of 102 cm(-1) between these signals suggests that acetate acts preferentially as a bidentate rather than as a bridging ligand between two titanium atoms. The use of acetic acid as modifier allows the control of both the degree of condensation and oligomerization of the precursor and leads to the preferential crystallization of TiO(2) in the anatase phase. A possible reaction pathway toward the formation of anatase is proposed on the basis of the intermediate species present in a 1:1 Ti(O(i)Pr)(4):CH(3)COOH molar system in which esterification reactions that introduce H(2)O into the reaction mixture were seen to be negligible. The Rietveld refinement and TEM analysis revealed that the powder is composed of isotropic anatase nanocrystallites.

Native crystal structure of a nitric oxide-releasing lectin from the seeds of Canavalia maritima

Here, we report the crystallographic study of a lectin from Canavalia maritima seeds (ConM) and its relaxant activity on vascular smooth muscle, to provide new insights into the understanding of structure/function relationships of this class of proteins. ConM was crystallized and its structure determined by standard molecular replacement techniques. The amino acid residues, previously suggested incorrectly by manual sequencing, have now been determined as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196, and S202. Analysis of the structure indicated a dimer in the asymmetric unit, two metal binding sites per monomer, and loops involved in the molecular oligomerization. These confer 98% similarity between ConM and other previously described lectins, derived from Canavalia ensiformis and Canavalia brasiliensis. Our functional data indicate that ConM exerts a concentration-dependent relaxant action on isolated aortic rings that probably occurs via an interaction with a specific lectin-binding site on the endothelium, resulting in a release of nitric oxide. (C) 2005 Elsevier B.V. All rights reserved.