Crystallization, data collection and phasing of two digestive lysozymes from Musca domestica

Lysozymes are mostly known for their defensive role against bacteria, but in several animals lysozymes have a digestive function. Here, the initial crystallographic characterization of two digestive lysozymes from Musca domestica are presented. The proteins were crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate or PEG/2-propanol as the precipitant. X-ray diffraction data were collected to a maximum resolution of 1.9 angstrom using synchrotron radiation. The lysozyme 1 and 2 crystals belong to the monoclinic space group P2(1) (unit-cell parameters a = 36.52, b = 79.44, c = 45.20 angstrom, beta = 102.97 degrees) and the orthorhombic space group P2(1)2(1)2 (unit-cell parameters a = 73.90, b = 96.40, c = 33.27 angstrom), respectively. The crystal structures were solved by molecular replacement and structure refinement is in progress.62875075

Cloning, Overexpression, Purification and Preliminary Characterization of Human Septin 8

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Mammalian septins comprise a family of 14 genes that encode GTP-binding proteins involved in important cellular processes such as cytokinesis and exocytosis. Expression of three different constructs encoding human septin 8 were analyzed and the results show that SEPT8GC, a clone expressing the conserved domain plus C-terminal domain of human septin 8 yields the highest amount of recombinant protein. This protein was purified by affinity chromatography followed by a gel filtration chromatography. CD spectrum of SEPT8GC is characteristic of folded proteins and it presents a transition profile with a T (m) of 54 A degrees C. Fluorescence emission spectra, analytic gel filtration and DLS reflect the sample oligomeric heterogeneity with the predominance of dimers in solution. Homology models indicate clearly that the preferred dimer interface is the one comprising the GTP binding site.295328335Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Associacao Brasileira de Tecnologia de Luz Sincrotron (ABTLuS)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP [2005/05149-6, 1998/14138-2

The acidic domain of hnRNPQ (NSAP1) has structural similarity to Barstar and binds to Apobec1

Apobecl edits the ApoB mRNA by deaminating nucleotide C-6666, which results in a codon change from Glutamate to stop, and subsequent expression of a truncated protein. Apobecl is regulated by ACF (Apobecl complementation factor) and hnRNPQ, which contains an N-terminal 'acidic domain' (AcD) of unknown function, three RNA recognition motifs, and an Arg/Gly-rich region. Here, we modeled the structure of AcD using the bacterial protein Barstar as a template. Furthermore, we demonstrated by in vitro pull-down assays that 6xHis-AcD alone is able to interact with GST-Apobecl. Finally, we performed in silico phosphorylation of AcD and molecular dynamics studies, which indicate conformational changes in the phosphorylated form. The results of the latter studies were confirmed by in vitro phosphorylation of 6xHis-AcD by protein kinase C, mass spectrometry, and spectroscopic analyses. Our data suggest hnRNPQ interactions via its AcD with Apobecl and that this interaction is regulated by the AcD phosphorylation. (c) 2006 Elsevier Inc. All rights reserved.350228829

The crystal structure of a lysozyme c from housefly Musca domestica, the first structure of a digestive lysozyme

Lysozymes from family 22 of glycoside hydrolases are usually part of the defense system against bacteria. However in ruminant art-iodactyls and saprophagous insects, lysozymes are involved in the digestion of bacteria. Here, we report the first crystallographic structure of a digestive lysozyme in its native and complexed forms, the structure of lysozyme I from Musca domestica larvae midgut (MdL1). Structural and biochemical data presented for MdL1 are analyzed in light of digestive lysozymes' traits. The structural core is similar, but a careful analysis of a structural alignment generated with other lysozymes c reveals that significant differences occur in coil regions. The loop from MdL1 defined by residues 98-100 has one deletion previous to residue Gln 100, which leads to a less exposed conformation and might justify the resistance to proteolysis observed for MdL1. In addition, G1n100 is directly involved in a few hydrogen bonds to the ligand in a yet unobserved substrate binding mode. The pK(a)s of the MdL1 catalytic residues (Glu32 and Asp50) are lower (6.40 and 3.09, respectively) than those from Gallus gallus egg lysozyme (GgL, hen egg white lysozyme-HEWL) (6.61 and 3.85, respectively). A unique feature of MdL1 is a hydrogen bond between Thr107 O gamma and Glu32 carboxylate group, which combined with the presence of Ser106 contributes to decrease the pK(a) of Glu32. Furthermore, in MdL1 the presence of Asn46 preventing the occurrence of an electrostatic repulsion with Asp50 and the increment in the solvent exposition of Asp50 due to Pro42 insertion contribute to reduce the pK(a) of Asp50. These structural elements affecting the pK(a)s of the catalytic residues should contribute to the acidic pH optimum presented by MdL1. (c) 2007 Elsevier Inc. All rights reserved.1601839

Expression and purification of a small heat shock protein from the plant pathogen Xylella fastidiosa

The small heat shock proteins (smHSPs) belong to a family of proteins that function as molecular chaperones by preventing protein aggregation and are also known to contain a conserved region termed a-crystallin domain. Here, we report the expression, purification, and partial characterization of a novel smHSP (HSP17.9) from the phytopathogen Xylella fastidiosa, causal agent of the citrus variegated chlorosis (CVC). The gene was cloned into a pET32-Xa/LIC vector to over-express the protein coupled with fusion tags in Escherichia coli BL21(DE3). The expressed HSP17.9 was purified by immobilized metal affinity chromatography (IMAC) and had its identity determined by mass spectrometry (MALDI-TOF). The correct folding of the purified recombinant protein was verified by circular dichroism spectroscopy. Finally, the HSP17.9 protein also proved to efficiently prevent induced aggregation of insulin, strongly indicating a chaperone-like activity. (C) 2003 Elsevier Inc. All rights reserved.33229730

The intrinsic antimicrobial activity of citric acid-coated manganese ferrite nanoparticles is enhanced after conjugation with the antifungal peptide Cm-p5

Carlos Lopez-Abarrategui,1 Viviana Figueroa-Espi,2 Maria B Lugo-Alvarez,1 Caroline D Pereira,3 Hilda Garay,4 João ARG Barbosa,5 Rosana Falcão,6 Linnavel Jiménez-Hernández,2 Osvaldo Estévez-Hernández,2,7 Edilso Reguera,8 Octavio L Franco,3,9 Simoni C Dias,3 Anselmo J Otero-Gonzalez1 1Faculty of Biology, Center for Protein Studies, 2Lab of Structural Analysis, Institute of Materials Science and Technology, Havana University, La Habana, Havana, Cuba; 3Center for Biochemical and Proteomics Analyses, Catholic University of Brasilia, Brasilia, Brazil; 4Laboratory of Peptide Analysis and Synthesis, Center of Genetic Engineering and Biotechnology, La Habana, Havana, Cuba; 5Department of Cellular Biology, Laboratory of Biophysics, Institute of Biological Science, University of Brasilia, 6Brazilian Agricultural Research Corporation (EMBRAPA), Center of Genetic Resources and Biotechnology (CENARGEN), Brasilia DF, Brazil; 7Instituto de Ciencia y Tecnología de Materiales (IMRE), Universidad de La Habana, Cuba; 8Research Center for Applied Science and Advanced Technology (CICATA), National Polytechnic Institute (IPN), Lagaria Unit, Mexico DF, Mexico; 9S-Inova Biotech, Post-Graduate in Biotechnology, Universidade Catolica Dom Bosco, Campo Grande, Brazil Abstract: Diseases caused by bacterial and fungal pathogens are among the major health problems in the world. Newer antimicrobial therapies based on novel molecules urgently need to be developed, and this includes the antimicrobial peptides. In spite of the potential of antimicrobial peptides, very few of them were able to be successfully developed into therapeutics. The major problems they present are molecule stability, toxicity in host cells, and production costs. A novel strategy to overcome these obstacles is conjugation to nanomaterial preparations. The antimicrobial activity of different types of nanoparticles has been previously demonstrated. Specifically, magnetic nanoparticles have been widely studied in biomedicine due to their physicochemical properties. The citric acid-modified manganese ferrite nanoparticles used in this study were characterized by high-resolution transmission electron microscopy, which confirmed the formation of nanocrystals of approximately 5 nm diameter. These nanoparticles were able to inhibit Candida albicans growth in vitro. The minimal inhibitory concentration was 250 µg/mL. However, the nanoparticles were not capable of inhibiting Gram-negative bacteria (Escherichia coli) or Gram-positive bacteria (Staphylococcus aureus). Finally, an antifungal peptide (Cm-p5) from the sea animal Cenchritis muricatus (Gastropoda: Littorinidae) was conjugated to the modified manganese ferrite nanoparticles. The antifungal activity of the conjugated nanoparticles was higher than their bulk counterparts, showing a minimal inhibitory concentration of 100 µg/mL. This conjugate proved to be nontoxic to a macrophage cell line at concentrations that showed antimicrobial activity. Keywords: nanoparticles, conjugation, antifungal, Cm-p5 peptid

Characterization of a novel N-acetylneuraminic acid lyase favoring industrial N-acetylneuraminic acid synthesis process

N-Acetylneuraminic acid lyase (NAL, E.C. number 4.1.3.3) is a Class I aldolase that catalyzes the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) from pyruvate and N-acetyl-D-mannosamine (ManNAc). Due to the equilibrium favoring Neu5Ac cleavage, the enzyme catalyzes the rate-limiting step of two biocatalytic reactions producing Neu5Ac in industry. We report the biochemical characterization of a novel NAL from a “GRAS” (General recognized as safe) strain C. glutamicum ATCC 13032 (CgNal). Compared to all previously reported NALs, CgNal exhibited the lowest kcat/Km value for Neu5Ac and highest kcat/Km values for ManNAc and pyruvate, which makes CgNal favor Neu5Ac synthesis the most. The recombinant CgNal reached the highest expression level (480 mg/L culture), and the highest reported yield of Neu5Ac was achieved (194 g/L, 0.63 M). All these unique properties make CgNal a promising biocatalyst for industrial Neu5Ac biosynthesis. Additionally, although showing the best Neu5Ac synthesis activity among the NAL family, CgNal is more related to dihydrodipicolinate synthase (DHDPS) by phylogenetic analysis. The activities of CgNal towards both NAL's and DHDPS' substrates are fairly high, which indicates CgNal a bi-functional enzyme. The sequence analysis suggests that CgNal might have adopted a unique set of residues for substrates recognition