Effect of dynamic high pressure on functional and structural properties of bovine serum albumin

Dynamic high pressure (DHP) has been investigated as an innovative suitable method to induce protein modifications. This work evaluated the effect of DHP (up to three passes at 100, 150 and 200 MPa, with an inlet temperature of 20 °C) on functional and structural properties of bovine serum albumin (BSA). Results indicated that DHP process applied up to an energy limit of 100 MPa increased the protein foaming capacity (FC) (p < 0.05 - increase up to 63% after 1 pass at 100 MPa) and the utilization of multiple passes at high pressure promoted a reduction in this property (p < 0.05 - reduction up to 31.6% after 3 passes at 200 MPa). Similar results were observed for sulfhydryl group, indicating an influence of free thiol groups on FC. Complementarily, DHP process promoted an increase of proteins particles size, suggesting a new rearrangement of their conformational structure. DHP did not affect tryptophan microenvironment in BSA; however, this process induced the rearrangement of secondary structure elements. In the first cycle, the pressure increase resulted in a loss of secondary structure, while in the second and third cycles the DHP process resulted in the gain of secondary structure elements. These results indicated that the second and third passes triggered a molecular rearrangement of the protein structure, giving rise to a novel and more stable conformational state. This conclusion was also supported by thermal unfolding studies (melting temperature reduction from 67.5 to 54.6 °C after 1 pass at 200 MPa), in which the additional cycles of DHP caused the occurrence of an initial denaturation at high temperatures, compared to the first cycle

Crystal structure of Jararacussin-I: The highly negatively charged catalytic interface contributes to macromolecular selectivity in snake venom thrombin-like enzymes

Submitted by Luciane Willcox ([email protected]) on 2016-09-02T18:33:57Z No. of bitstreams: 1 Crystal structure of Jararacussin-I.pdf: 356808 bytes, checksum: cd01e76570df32e71244d77604645768 (MD5)Approved for entry into archive by Luciane Willcox ([email protected]) on 2016-09-02T19:10:05Z (GMT) No. of bitstreams: 1 Crystal structure of Jararacussin-I.pdf: 356808 bytes, checksum: cd01e76570df32e71244d77604645768 (MD5)Made available in DSpace on 2016-09-02T19:10:05Z (GMT). No. of bitstreams: 1 Crystal structure of Jararacussin-I.pdf: 356808 bytes, checksum: cd01e76570df32e71244d77604645768 (MD5) Previous issue date: 2012-11-08FAPESP, CNPq, TWAS, DAAD, CAPESUniversidade Estadual Paulista. Instituto de Biociências Letras e Ciências Exatas. Departamento de Física. Centro Multiusuário de Inovação Biomolecular, São Jose do Rio Preto, SP, Brasil.Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Biociências. Campinas, SP, Brasil / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Universidade Estadual Paulista. Instituto de Biociências Letras e Ciências Exatas. Departamento de Física. Centro Multiusuário de Inovação Biomolecular, São Jose do Rio Preto, SP, Brasil.Universidade Estadual Paulista. Instituto de Biociências Letras e Ciências Exatas. Departamento de Física. Centro Multiusuário de Inovação Biomolecular, São Jose do Rio Preto, SP, Brasil.Universidade Estadual Paulista. Instituto de Biociências Letras e Ciências Exatas. Departamento de Física. Centro Multiusuário de Inovação Biomolecular, São Jose do Rio Preto, SP, Brasil.Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Biociências. Campinas, SP, Brasil.Universidade Estadual Paulista. Instituto de Biociências Letras e Ciências Exatas. Departamento de Física. Centro Multiusuário de Inovação Biomolecular, São Jose do Rio Preto, SP, Brasil.Snake venom serine proteinases (SVSPs) are hemostatically active toxins that perturb the maintenance and regulation of both the blood coagulation cascade and fibrinolytic feedback system at specific points, and hence, are widely used as tools in pharmacological and clinical diagnosis. The crystal structure of a thrombin-like enzyme (TLE) from Bothrops jararacussu venom (Jararacussin-I) was determined at 2.48 Å resolution. This is the first crystal structure of a TLE and allows structural comparisons with both the Agkistrodon contortrix contortrix Protein C Activator and the Trimeresurus stejnegeri plasminogen activator. Despite the highly conserved overall fold, significant differences in the amino acid compositions and three-dimensional conformations of the loops surrounding the active site significantly alter the molecular topography and charge distribution profile of the catalytic interface. In contrast to other SVSPs, the catalytic interface of Jararacussin-I is highly negatively charged, which contributes to its unique macromolecular selectivity

Production, partial characterization, and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus Myceliophthora sp.

Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50A degrees C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Crystal structure of mature 2S albumin from Moringa oleifera seeds

2S albumins, the seed storage proteins, are the primary sources of carbon and nitrogen and are involved in plant defense. The mature form of Moringa oleifera (M. oleifera), a chitin binding protein isoform 3-1 (mMo-CBP3-1) a thermostable antifungal, antibacterial, flocculating 2S albumin is widely used for the treatment of water and is potentially interesting for the development of both antifungal drugs and transgenic crops. The crystal structure of mMo-CBP3-1 determined at 1.7 Å resolution demonstrated that it is comprised of two proteolytically processed α-helical chains, stabilized by four disulfide bridges that is stable, resistant to pH changes and has a melting temperature (TM) of approximately 98 °C. The surface arginines and the polyglutamine motif are the key structural factors for the observed flocculating, antibacterial and antifungal activities. This represents the first crystal structure of a 2S albumin and the model of the pro-protein indicates the structural changes that occur upon formation of mMo-CBP3-1 and determines the structural motif and charge distribution patterns for the diverse observed activities.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científco e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Structural basis for glucose tolerance in GH1 b-glucosidases

Submitted by Tatiana Souza ([email protected]) on 2014-10-24T14:05:46Z No. of bitstreams: 1 J. Biol. Chem.-1997-Nare-13883-91.pdf: 1511856 bytes, checksum: 1819e3063bfd29935a7afcb91b0aa294 (MD5)Approved for entry into archive by Tatiana Souza ([email protected]) on 2014-11-25T17:03:08Z (GMT) No. of bitstreams: 1 J. Biol. Chem.-1997-Nare-13883-91.pdf: 1511856 bytes, checksum: 1819e3063bfd29935a7afcb91b0aa294 (MD5)Made available in DSpace on 2014-11-25T17:03:08Z (GMT). No. of bitstreams: 1 J. Biol. Chem.-1997-Nare-13883-91.pdf: 1511856 bytes, checksum: 1819e3063bfd29935a7afcb91b0aa294 (MD5) Previous issue date: 2014Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Biociências. Campinas, SP, Brasil. Campinas, SP, Brasil.Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Biociências. Campinas, SP, Brasil. Campinas, SP, Brasil / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Universidade de São Paulo. Faculdade de Filosofia, Ciências e Letras. Departamento de Química. Ribeirão Preto, SP, Brasil.Universidade Estadual de Campinas. Instituto de Química. Departamento de Química Orgânica. Campinas, SP, Brasil.Universidade de São Paulo. Faculdade de Filosofia, Ciências e Letras. Departamento de Química. Ribeirão Preto, SP, Brasil / Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de de Ciência e Tecnologia do Bioetanol. Campinas, SP, Brasil.Universidade de São Paulo. Faculdade de Filosofia, Ciências e Letras. Departamento de Química. Ribeirão Preto, SP, Brasil / Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de de Ciência e Tecnologia do Bioetanol. Campinas, SP, Brasil.Universidade de São Paulo. Faculdade de Filosofia, Ciências e Letras. Departamento de Biologia. Ribeirão Preto, SP, Brasil.Universidade de São Paulo. Faculdade de Filosofia, Ciências e Letras. Departamento de Química. Ribeirão Preto, SP, Brasil.Centro Nacional de Pesquisa em Energia e Materiais. Laboratório Nacional de Biociências. Campinas, SP, Brasil. Campinas, SP, Brasil.Product inhibition of β-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification. Remarkably, some β-glucosidases belonging to the GH1 family exhibit unusual properties, being tolerant to, or even stimulated by, high glucose concentrations. However, the structural basis for the glucose tolerance and stimulation of BGs is still elusive. To address this issue, the first crystal structure of a fungal β-glucosidase stimulated by glucose was solved in native and glucose-complexed forms, revealing that the shape and electrostatic properties of the entrance to the active site, including the +2 subsite, determine glucose tolerance. The aromatic Trp168 and the aliphatic Leu173 are conserved in glucose-tolerant GH1 enzymes and contribute to relieving enzyme inhibition by imposing constraints at the +2 subsite that limit the access of glucose to the -1 subsite. The GH1 family β-glucosidases are tenfold to 1000-fold more glucose tolerant than GH3 BGs, and comparative structural analysis shows a clear correlation between active-site accessibility and glucose tolerance. The active site of GH1 BGs is located in a deep and narrow cavity, which is in contrast to the shallow pocket in the GH3 family BGs. These findings shed light on the molecular basis for glucose tolerance and indicate that GH1 BGs are more suitable than GH3 BGs for biotechnological applications involving plant cell-wall saccharification

Data collection and refinement statistics.

<p>Data collection and refinement statistics.</p

Effect of pH and temperature on the catalytic activity of AfmE1.

<p>(A) Determination of optimum pH. The hydrolytic activity was measured at different pHs at 40°C for 10 min. (B) Determination of optimum temperature. The hydrolytic activity was measured at temperatures ranging from 20 to 70°C. (C) Thermal stability assay. The enzyme was incubated at 45, 50 and 55°C for up to 4 h and residual activity was determined under the optimal reaction conditions. Error bars represent the standard deviation.</p

AfmE1 substrate-binding cleft.

<p>(A) Molecular surface of AfmE1 with its stacking residue Trp272 in subsite +3 represented as sticks with carbon atoms in green. The stacking residue Tyr369 in the corresponding region of CelA from <i>C</i>. <i>thermocellum</i> is similarly represented in magenta to evidence the differences in the subsite +3 configuration of these enzymes. The region containing the catalytic residues is highlighted in yellow. The substrate molecules, represented as deduced from the complex of CelA with cellopentaose (white) and cellotriose (blue) (PDB code 1KWF), as well as of BcsZ with cellopentaose (orange) (PDB code 3QXQ), are shown as sticks to indicate the position of the subsites. (B) AfmE1 substrate-binding cleft highlighting the catalytic (yellow) and the glucosyl-stacking residues (green) in its six subsites (dashed lines). The corresponding stacking residues of the proteins CMCax, BcsZ and CelA are shown in cyan, orange and magenta, respectively. The position of the glucosyl residues (blue) occupying the six subsites was predicted from the complex CelA-substrate [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176550#pone.0176550.ref044" target="_blank">44</a>].</p

AfmE1 mode of action.

<p>Thin-layer chromatography analysis of degradation products derived from AfmE1-mediated hydrolysis of different cello-oligosaccharides. (A) cellobiose, cellotriose and cellotetraose; (B) cellopentaose and (C) cellohexaose. The first line of each panel corresponds to a mixture of the indicated standards. CZE electropherograms of the APTS-labeled products of cellopentaose (D) and cellohexaose (E) hydrolysis after 0, 2 and 4 h of incubation with AfmE1. CZE electropherograms of the APTS-labeled products from AfmE1-mediated hydrolysis of β-glucan (F) and CMC (G). The labeled cello-oligosaccharides are indicated, as inferred from a parallel run of a standard mixture. For all the analyses, control reactions were carried out in the absence of AfmE1 and run in parallel.</p