Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 30
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Chem Inf Model ; 2020 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-31895561

RESUMO

Trypsin-like serine proteases are a group of homologous enzymes which exert multiple roles in both vertebrate and invertebrate organisms. Key properties of these enzymes include their activation from an inactive zymogen form to their active form by cleavage of residues in their N-terminus, the presence of a conserved catalytic triad of residues, and the existence of different patterns of substrate selectivity for residue cleavage between the various members of this protein family. In this article, we apply the decomposition of residue coevolution networks computational method to find sets of residues related to some of these key properties, especially to zymogen activation. Positive selection detection, normal modes analysis, and the calculation of thermal couplings between the bovine trypsinogen and bovine trypsin structures residues yielded further information for understanding the zymogen activation process and highlighted the importance of some of the coevolved set residues during these transitions.

2.
Mol Immunol ; 112: 151-162, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31108423

RESUMO

Pb27 antigen is an interesting alternative to immunological diagnosis of Paracoccidioidomycosis (PCM) and has demonstrated to be protective in experimental PCM. Its tertiary structure and possible function remained unknown till now. To study Pb27 at the atomic level, the recombinant protein was expressed in Escherichia coli BL21(DE3), purified, and its three-dimensional structure was solved by X-ray crystallography. Based on this structure, we performed a residue correlation analysis and in silico ligand search assays to address a possible biological function to Pb27. We identified Pb27 as a member of the extensive nucleotidyltransferase superfamily. The protein has an αßαßαß topology with two domains (N- and C-terminal domains) and adopts a monomeric form as its biological unit in solution. Structural comparisons with similar members of the superfamily clearly indicate Pb27 C-terminal domain is singular and may play an important role in its biological function. Bioinformatics analysis suggested that Pb27 might bind to ATP and CTP. This suggestion is corroborated by the fact that a magnesium cation is coordinated by two aspartic acid residues present at the active site (between N- and C-terminal domains), as evidenced by X-ray diffraction data. Besides, NMR assays (1H-15N HSQC spectra) confirmed the binding of CTP to Pb27, demonstrating for the first time an interaction between a nucleotide and this protein. Moreover, we evaluated the reactivity of sera from patients with Paracoccidioides brasiliensis infection against the recombinant form of Pb27 and showed that it was recognized by sera from infected and treated patients. Predicted B and T cell epitopes were synthesized and further evaluated against sera of PCM patients, providing information of the most reactive peptides in Pb27 primary structure which interact with specific Pb27 antibodies.


Assuntos
Proteínas Fúngicas/imunologia , Nucleotidiltransferases/imunologia , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Trifosfato de Adenosina/imunologia , Adolescente , Adulto , Idoso , Sequência de Aminoácidos , Citidina Trifosfato/imunologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Escherichia coli/imunologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Recombinantes/imunologia , Adulto Jovem
3.
Bioinformatics ; 35(9): 1478-1485, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-30295749

RESUMO

MOTIVATION: Computational studies of molecular evolution are usually performed from a multiple alignment of homologous sequences, on which sequences resulting from a common ancestor are aligned so that equivalent residues are placed in the same position. Residues frequency patterns of a full alignment or from a subset of its sequences can be highly useful for suggesting positions under selection. Most methods mapping co-evolving or specificity determinant sites are focused on positions, however, they do not consider the case for residues that are specificity determinants in one subclass, but variable in others. In addition, many methods are impractical for very large alignments, such as those obtained from Pfam, or require a priori information of the subclasses to be analyzed. RESULTS: In this paper we apply the complex networks theory, widely used to analyze co-affiliation systems in the social and ecological contexts, to map groups of functional related residues. This methodology was initially evaluated in simulated environments and then applied to four different protein families datasets, in which several specificity determinant sets and functional motifs were successfully detected. AVAILABILITY AND IMPLEMENTATION: The algorithms and datasets used in the development of this project are available on http://www.biocomp.icb.ufmg.br/biocomp/software-and-databases/networkstats/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

4.
Biochem Biophys Res Commun ; 506(4): 826-832, 2018 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-30389137

RESUMO

Voltage-gated sodium channels play important roles in human physiology. However, their complexity hinders the understanding of their physiology and pathology at atomic level. We took advantage of the structural reports of similar channels obtained by cryo-EM (EeNav1.4, and NavPaS), and constructed models of human Nav1.4 channels at closed and open states. The open-state model is very similar to the recently published cryo-EM structure of hNav1.4. The comparison of both models shows shifts of the voltage sensors (VS) of DIII and DIV. The activated position of VS-DII in the closed model was demonstrated by Ts1 docking, thereby confirming the requirement that VS-DI, VS-DII and VS-DIII must be activated for the channel to open. The interactions observed with VS-DIII suggest a stepwise, yet fast, transition from resting to activated state. These models provide structural insights on the closed-open transition of the channel.


Assuntos
Ativação do Canal Iônico , Modelos Biológicos , Músculo Esquelético/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.4/química , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Humanos , Simulação de Acoplamento Molecular
5.
Gene ; 666: 58-63, 2018 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-29733970

RESUMO

OBJECTIVE: Thyroxine-binding globulin (TBG) is the major human thyroid hormone transport protein, encoded by the SERPINA7 gene (Xq22.2). We aim to investigate the molecular basis of partial TBG deficiency (TBG-PD) in a female, by evaluating the X-chromosome inactivation pattern as well as the mutant protein structural modeling. DESIGN AND METHODS: Sequencing of the coding region of the SERPINA7 gene was performed in a female with a TBG-PD phenotype and her first-degree relatives. The proband presented with low serum levels of total T3 (TT3) and total T4 (TT4), serum TSH level of 5.4 µUI/mL (normal range, 0.35-5.5), and serum TBG level of 5.5 mg/L (normal range, 13.6-27.2). X-chromosome inactivation pattern was evaluated by methylation analysis of the androgen receptor gene (Xq11.2). Structural analysis of the SERPIN family was performed using Pymol and Areaimol, and PFSTATS for conservation analysis and family-wide investigation of equivalent positions in human homologs. Modeller was used for point mutation structural modeling. RESULTS: A novel missense SERPINA7 mutation (p.R35W; c.163C > T) was found in heterozygosity in the proband, and in hemizygosity in her affected siblings. The proband X-chromosome inactivation ratio was 20:80. The substitution of an arginine by a tryptophan is predicted to disrupt the protein surface and main electrostatic interactions. Tryptophans are extremely rare (0.1%) in this position. CONCLUSIONS: We report a new SERPINA7 variant associated with TBG-PD in three siblings. We named this variant TBG-Brasilia. The X-chromosome inactivation pattern may have accounted for the rare phenotypic expression in a female. The hydrophobic nature of the mutant is predicted to create an apolar patch at the surface, which results in protein aggregation and/or misfolding, potentially responsible for thyroid hormone transport defect.


Assuntos
Doenças Genéticas Ligadas ao Cromossomo X/genética , Globulina de Ligação a Tiroxina/deficiência , Adulto , Sequência de Bases , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto , Linhagem , Mutação Puntual , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Globulina de Ligação a Tiroxina/química , Globulina de Ligação a Tiroxina/genética , Inativação do Cromossomo X
6.
Toxicon ; 146: 50-60, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29608922

RESUMO

Phospholipases A2 (PLA2s) comprise a superfamily of glycerophospholipids hydrolyzing enzymes present in many organisms in nature, whose catalytic activity was majorly unveiled by analysis of snake venoms. The latter have pharmaceutical and biotechnological interests and can be divided into different functional sub-classes. Our goal was to identify important residues and their relation to the functional and class-specific characteristics in the PLA2s family with special emphasis on snake venom PLA2s (svPLA2s). We identified such residues by conservation analysis and decomposition of residue coevolution networks (DRCN), annotated the results based on the available literature on PLA2s, structural analysis and molecular dynamics simulations, and related the results to the phylogenetic distribution of these proteins. A filtered alignment of PLA2s revealed 14 highly conserved positions and 3 sets of coevolved residues, which were annotated according to their structural or functional role. These residues are mostly involved in ligand binding and catalysis, calcium-binding, the formation of disulfide bridges and a hydrophobic cluster close to the binding site. An independent validation of the inference of structure-function relationships from our co-evolution analysis on the svPLA2s family was obtained by the analysis of the pattern of selection acting on the Viperidae and Elapidae lineages. Additionally, a molecular dynamics simulation on the Lys49 PLA2 from Agkistrodon contortrix laticinctus was carried out to further investigate the correlation of the Lys49-Glu69 pair. Our results suggest this configuration can result in a novel conformation where the binding cavity collapses due to the approximation of two loops caused by a strong salt bridge between Glu69 and Arg34. Finally, phylogenetic analysis indicated a correlation between the presence of residues in the coevolved sets found in this analysis and the clade localization. The results provide a guide for important positions in the family of PLA2s, and potential new objects of investigation.


Assuntos
Fosfolipases A2/química , Venenos de Serpentes/enzimologia , Relação Estrutura-Atividade , Agkistrodon , Animais , Elapidae , Simulação de Dinâmica Molecular , Filogenia , Estrutura Terciária de Proteína , Venenos de Serpentes/química , Viperidae
7.
J Comput Biol ; 25(5): 480-486, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29481292

RESUMO

PFstats is a software developed for the extraction of useful information from protein multiple sequence alignments. By analyzing positional conservation and residue coevolution networks, the software allows the identification of structurally and functionally important residue groups and the discovery of probable functional subclasses. Furthermore, it contains tools for the identification of the possible biological significance of these findings. PFstats contains methods for maximizing the significance of alignments through filtering and weighting, residue conservation and coevolution analysis, automatic UniprotKb queries for residue-position annotation and many possible data visualization methods.


Assuntos
Aspartato Carbamoiltransferase/metabolismo , Citrato (si)-Sintase/metabolismo , Família Multigênica , Ornitina Carbamoiltransferase/metabolismo , Mapas de Interação de Proteínas , Análise de Sequência de Proteína/métodos , Software , Aspartato Carbamoiltransferase/química , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Citrato (si)-Sintase/química , Biologia Computacional , Bases de Dados de Proteínas , Humanos , Ornitina Carbamoiltransferase/química
8.
Cell Biol Int ; 42(6): 630-642, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29160602

RESUMO

Saccharomyces cerevisiae mitoribosomes are specialized in the translation of a few number of highly hydrophobic membrane proteins, components of the oxidative phosphorylation system. Mitochondrial characteristics, such as the membrane system and its redox state driven mitoribosomes evolution through great diversion from their bacterial and cytosolic counterparts. Therefore, mitoribosome presents a considerable number of mitochondrial-specific proteins, as well as new protein extensions. In this work we characterize temperature sensitive mutants of the subunit bL34 present in the 54S large subunit. Although bL34 has bacterial homologs, in yeast it has a long 65 aminoacids mitochondrial N-terminal addressing sequence, here we demonstrate that it can be replaced by the mitochondrial addressing sequence of Neurospora crassa ATP9 gene. The bL34 temperature sensitive mutants present lowered translation of mitochondrial COX1 and COX3, which resulted in reduced cytochrome c oxidase activity and respiratory growth deficiency. The sedimentation properties of bL34 in sucrose gradients suggest that similarly to its bacterial homolog, bL34 is also a later participant in the process of mitoribosome biogenesis.


Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Ribossomos Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Complexo IV da Cadeia de Transporte de Elétrons/genética , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Mutagênese Sítio-Dirigida , Biossíntese de Proteínas , Proteínas RGS/genética , Proteínas RGS/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência
9.
Sci Rep ; 7(1): 11231, 2017 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-28894204

RESUMO

Bacteria are essential in arsenic cycling. However, few studies have addressed 16S rRNA and arsenic-related functional gene diversity in long-term arsenic-contaminated tropical sediment. Here, using culture-based, metagenomic and computational approaches, we describe the diversity of bacteria, genes and enzymes involved in AsIII and AsV transformation in freshwater sediment and in anaerobic AsIII- and AsV-enrichment cultures (ECs). The taxonomic profile reveals significant differences among the communities. Arcobacter, Dechloromonas, Sedimentibacter and Clostridium thermopalmarium were exclusively found in ECs, whereas Anaerobacillus was restricted to AsV-EC. Novel taxa that are both AsV-reducers and AsIII-oxidizers were identified: Dechloromonas, Acidovorax facilis, A. delafieldii, Aquabacterium, Shewanella, C. thermopalmarium and Macellibacteroides fermentans. Phylogenic discrepancies were revealed among the aioA, arsC and arrA genes and those of other species, indicating horizontal gene transfer. ArsC and AioA have sets of amino acids that can be used to assess their functional and structural integrity and familial subgroups. The positions required for AsV reduction are conserved, suggesting strong selective pressure for maintaining the functionality of ArsC. Altogether, these findings highlight the role of freshwater sediment bacteria in arsenic mobility, and the untapped diversity of dissimilatory arsenate-reducing and arsenate-resistant bacteria, which might contribute to arsenic toxicity in aquatic environments.


Assuntos
Arsênico/metabolismo , Bactérias/classificação , Água Doce/microbiologia , Variação Genética , Sedimentos Geológicos/microbiologia , Redes e Vias Metabólicas/genética , Poluentes Químicos da Água/metabolismo , Anaerobiose , Bactérias/genética , Bactérias/isolamento & purificação , Biotransformação , Análise por Conglomerados , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Enzimas/genética , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA
10.
PLoS One ; 12(5): e0177090, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28472157

RESUMO

In Saccharomyces cerevisiae mitochondrial dysfunction induces retrograde signaling, a pathway of communication from mitochondria to the nucleus that promotes a metabolic remodeling to ensure sufficient biosynthetic precursors for replication. Rtg2p is a positive modulator of this pathway that is also required for cellular longevity. This protein belongs to the ASKHA superfamily, and contains a putative N-terminal ATP-binding domain, but there is no detailed structural and functional map of the residues in this domain that accounts for their contribution to retrograde signaling and aging. Here we use Decomposition of Residue Correlation Networks and site-directed mutagenesis to identify Rtg2p structural determinants of retrograde signaling and longevity. We found that most of the residues involved in retrograde signaling surround the ATP-binding loops, and that Rtg2p N-terminus is divided in three regions whose mutants have different aging phenotypes. We also identified E137, D158 and S163 as possible residues involved in stabilization of ATP at the active site. The mutants shown here may be used to map other Rtg2p activities that crosstalk to other pathways of the cell related to genomic stability and aging.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Modelos Moleculares , Mutação , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
11.
Biochem Biophys Res Commun ; 492(4): 565-571, 2017 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-28087275

RESUMO

Flaviviruses are responsible for serious diseases such as dengue, yellow fever, and zika fever. Their genomes encode a polyprotein which, after cleavage, results in three structural and seven non-structural proteins. Homologous proteins can be studied by conservation and coevolution analysis as detected in multiple sequence alignments, usually reporting positions which are strictly necessary for the structure and/or function of all members in a protein family or which are involved in a specific sub-class feature requiring the coevolution of residue sets. This study provides a complete conservation and coevolution analysis on all flaviviruses non-structural proteins, with results mapped on all well-annotated available sequences. A literature review on the residues found in the analysis enabled us to compile available information on their roles and distribution among different flaviviruses. Also, we provide the mapping of conserved and coevolved residues for all sequences currently in SwissProt as a supplementary material, so that particularities in different viruses can be easily analyzed.


Assuntos
Sequência Conservada/genética , Evolução Molecular , Flavivirus/genética , Genoma Viral/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Sequência de Aminoácidos/genética , Domínios Proteicos/genética , Relação Estrutura-Atividade
12.
Water Res ; 110: 27-37, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-27984803

RESUMO

Wastewater treatment plants (WWTPs) harbor bacteria and antimicrobial resistance genes, favoring gene exchange events and resistance dissemination. Here, a culture-based and metagenomic survey of qnrA, qnrB, qnrS, and aac(6')-Ib genes from raw sewage (RS) and activated sludge (AS) of a full-scale municipal WWTP was performed. A total of 96 bacterial isolates were recovered from nalidixic acid-enrichment cultures. Bacteria harboring the aac(6')-Ib gene predominated in RS, whereas qnrS-positive isolates were specific to AS. Novel qnrS- and aac(6')-Ib-cr positive species were identified: Morganella morganii, Providencia rettgeri, and Pseudomonas guangdongensis (qnrS), and Alcaligenes faecalis and P. rettgeri (aac(6')-Ib-cr). Analysis of qnrS and aac(6')-Ib sequences from isolates and clone libraries suggested that the diversity of qnrS is wider than that of aac(6')-Ib. A large number of amino acid mutations were observed in the QnrS and AAC(6')-Ib proteins at previously undetected positions, whose structural implications are not clear. An accumulation of mutations at the C72, Q73, L74, A75 and M76 positions of QnrS, and D181 of AAC(6')-Ib might be important for resistance. These findings add significant information on bacteria harboring qnrS and aac(6')-Ib genes, and the presence of novel mutations that may eventually emerge in clinical isolates.


Assuntos
Escherichia coli/isolamento & purificação , Esgotos , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Fluoroquinolonas , Testes de Sensibilidade Microbiana , Mutação , Plasmídeos/efeitos dos fármacos , Quinolonas
13.
Biochimie ; 119: 92-102, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26497406

RESUMO

Coenzyme Q (Q) is an isoprenylated benzoquinone electron carrier required for electronic transport in the mitochondrial respiratory chain, shuttling electrons from complexes I and II to complex III. Q synthesis requires proteins termed Coq (Coq1-Coq11). Coq7p is part of the multimeric complex involved in Q synthesis catalyzing the hydroxylation of demethoxy-Q6 (DMQ6), the last monooxygenase step in Q synthesis with a catalytic center containing a carboxylate-bridged di-iron at the active site of the enzyme. Here we indicate a group of Coq7p residues that modulate protein activity: D53, R57, V111 and S114. R57, V111 and S114 are very conserved residues; V111 and S114 are present in separated communities of amino acid correlation analysis. The coq7 double mutant V111G/S114A and S114E show respiratory deficiency at non permissive temperature, DMQ6 accumulation and lower content of Q6. Therefore we conclude that phosphomimetic S114E inhibit Coq7p activity, and propose that S114 phosphorylation is required to move a non-structured loop of 25 amino acids between helix 2 and 3, and that affects the di-iron coordination in Coq7p catalytic center.


Assuntos
Membranas Mitocondriais/enzimologia , Modelos Moleculares , Ferroproteínas não Heme/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Ubiquinona/biossíntese , Sequência de Aminoácidos , Substituição de Aminoácidos , Biocatálise , Sequência Conservada , Estabilidade Enzimática , Temperatura Alta/efeitos adversos , Hidroxilação , Membranas Mitocondriais/metabolismo , Mutagênese Sítio-Dirigida , Mutação , Ferroproteínas não Heme/química , Ferroproteínas não Heme/genética , Fosforilação , Filogenia , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência
14.
Fungal Genet Biol ; 60: 133-9, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23850602

RESUMO

Bacterial GatCAB amidotransferases are responsible for the transamidation of mischarged glutamyl-tRNA(Gln) into glutaminyl-tRNA(Gln). Mitochondria matrix also has a multienzymatic complex necessary for the transamidation of glutamyl-tRNA(Gln). Gtf1p, Her2p and Pet112p are the constituents of mitochondrial GatFAB amidotransferase complex. Her2p is subunit A of GatFAB complex, while Gtf1p is subunit F, a connector protein between Pet112p (subunit B) and Her2p. Here we evaluate through molecular modeling and amino acid correlation analysis the HER2 protein family. Localization studies indicated that Her2p is predominantly localized in the mitochondrial outer membrane, but it is also located in the mitochondrial matrix where together with Pet112p and Gtf1p constitutes the GatFAB complex. Finally, HER2 random mutagenesis unveiled important residues that provide thermo stability for the complex and are differently suppressed by overexpression of GTF1 or PET112. For instance, her2/ts11 mutant showed its fermentative growth impaired, and poorly rescued by GTF1 indicating that Her2p unknown function in the mitochondria outer membrane affects cell viability.


Assuntos
Aminoacil-tRNA Sintetases/genética , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Transferases de Grupos Nitrogenados/genética , Transferases de Grupos Nitrogenados/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transaminases/genética , Aminoacil-tRNA Sintetases/metabolismo , Sobrevivência Celular , Mapeamento Cromossômico , Retículo Endoplasmático/metabolismo , Glutamina/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Modelos Moleculares , Mutação , Aminoacil-RNA de Transferência/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Transaminases/metabolismo
15.
BMC Genomics ; 14 Suppl 6: S1, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24564869

RESUMO

BACKGROUND: Nuclear receptors (NRs) are transcription factors which bind small hormones, whose evolutionary history and the presence of different functional surfaces makes them an interesting target for a correlation based analysis. RESULTS: Correlation analysis of ligand binding domains shows that correlated residue subsets arise from the differences between functional sites in different nuclear receptor subfamilies. For the DNA binding domain, particularly, the analysis shows that the main source of correlation comes from residues that regulate hormone response element specificity, and one of the conserved residue sub-sets arises due to the presence of an unusual sequence for the DNA binding motif known as P-box in nematodes, suggesting the existence of different DBD-DNA specificities in nuclear receptors. CONCLUSIONS: We conclude that DNA specificity and functional surface specialization has independently driven nuclear receptor evolution, and suggest possible binding modes for the class of divergent nuclear receptors in nematodes.


Assuntos
Aminoácidos/metabolismo , Proteínas de Helminto/química , Proteínas de Helminto/metabolismo , Nematoides/metabolismo , Receptores Citoplasmáticos e Nucleares/química , Receptores Citoplasmáticos e Nucleares/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ligantes , Modelos Moleculares , Nematoides/genética , Ligação Proteica , Mapas de Interação de Proteínas , Estrutura Terciária de Proteína , Especificidade da Espécie
16.
PLoS One ; 6(12): e27786, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22205928

RESUMO

Correlated mutation analysis has a long history of interesting applications, mostly in the detection of contact pairs in protein structures. Based on previous observations that, if properly assessed, amino acid correlation data can also provide insights about functional sub-classes in a protein family, we provide a complete framework devoted to this purpose. An amino acid specific correlation measure is proposed, which can be used to build networks summarizing all correlation and anti-correlation patterns in a protein family. These networks can be submitted to community structure detection algorithms, resulting in subsets of correlated amino acids which can be further assessed by specific parameters and procedures that provide insight into the relationship between different communities, the individual importance of community members and the adherence of a given amino acid sequence to a given community. By applying this framework to three protein families with contrasting characteristics (the Fe/Mn-superoxide dismutases, the peroxidase-catalase family and the C-type lysozyme/α-lactalbumin family), we show how our method and the proposed parameters and procedures are related to biological characteristics observed in these protein families, highlighting their potential use in protein characterization and gene annotation.


Assuntos
Algoritmos , Aminoácidos , Biologia Computacional/métodos , Proteínas/química , Proteínas/metabolismo , Cálcio/metabolismo , Catalase/química , Catalase/metabolismo , Lactalbumina/química , Lactalbumina/metabolismo , Modelos Moleculares , Muramidase/química , Muramidase/metabolismo , Peroxidase/química , Peroxidase/metabolismo , Conformação Proteica , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo
17.
J Phys Chem B ; 115(24): 7940-9, 2011 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-21619042

RESUMO

Glycosyl hydrolases are enzymes capable of breaking the glycosidic linkage of polysaccharides and have considerable industrial and biotechnological applications. Driven by the later applications, it is frequently desirable that glycosyl hydrolases display stability and activity under extreme environment conditions, such as high temperatures and extreme pHs. Here, we present X-ray structure of the hyperthermophilic laminarinase from Rhodothermus marinus (RmLamR) determined at 1.95 Å resolution and molecular dynamics simulation studies aimed to comprehend the molecular basis for the thermal stability of this class of enzymes. As most thermostable proteins, RmLamR contains a relatively large number of salt bridges, which are not randomly distributed on the structure. On the contrary, they form clusters interconnecting ß-sheets of the catalytic domain. Not all salt bridges, however, are beneficial for the protein thermostability: the existence of charge-charge interactions permeating the hydrophobic core of the enzymes actually contributes to destabilize the structure by facilitating water penetration into hydrophobic cavities, as can be seen in the case of mesophilic enzymes. Furthermore, we demonstrate that the mobility of the side-chains is perturbed differently in each class of enzymes. The side-chains of loop residues surrounding the catalytic cleft in the mesophilic laminarinase gain mobility and obstruct the active site at high temperature. By contrast, thermophilic laminarinases preserve their active site flexibility, and the active-site cleft remains accessible for recognition of polysaccharide substrates even at high temperatures. The present results provide structural insights into the role played by salt-bridges and active site flexibility on protein thermal stability and may be relevant for other classes of proteins, particularly glycosyl hydrolases.


Assuntos
Celulases/química , Simulação de Dinâmica Molecular , Rhodothermus/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Dados de Sequência Molecular , Estabilidade Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Temperatura Ambiente
18.
J Biol Chem ; 285(41): 31731-41, 2010 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-20659897

RESUMO

Human transthyretin (TTR) is a homotetrameric protein involved in several amyloidoses. Zn(2+) enhances TTR aggregation in vitro, and is a component of ex vivo TTR amyloid fibrils. We report the first crystal structure of human TTR in complex with Zn(2+) at pH 4.6-7.5. All four structures reveal three tetra-coordinated Zn(2+)-binding sites (ZBS 1-3) per monomer, plus a fourth site (ZBS 4) involving amino acid residues from a symmetry-related tetramer that is not visible in solution by NMR. Zn(2+) binding perturbs loop E-α-helix-loop F, the region involved in holo-retinol-binding protein (holo-RBP) recognition, mainly at acidic pH; TTR affinity for holo-RBP decreases ∼5-fold in the presence of Zn(2+). Interestingly, this same region is disrupted in the crystal structure of the amyloidogenic intermediate of TTR formed at acidic pH in the absence of Zn(2+). HNCO and HNCA experiments performed in solution at pH 7.5 revealed that upon Zn(2+) binding, although the α-helix persists, there are perturbations in the resonances of the residues that flank this region, suggesting an increase in structural flexibility. While stability of the monomer of TTR decreases in the presence of Zn(2+), which is consistent with the tertiary structural perturbation provoked by Zn(2+) binding, tetramer stability is only marginally affected by Zn(2+). These data highlight structural and functional roles of Zn(2+) in TTR-related amyloidoses, as well as in holo-RBP recognition and vitamin A homeostasis.


Assuntos
Amiloidose , Pré-Albumina/química , Multimerização Proteica , Proteínas de Ligação ao Retinol/química , Zinco/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Pré-Albumina/metabolismo , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas de Ligação ao Retinol/metabolismo , Vitamina A/química , Vitamina A/metabolismo , Zinco/metabolismo
19.
J Struct Biol ; 170(3): 522-31, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20211733

RESUMO

Transthyretin (TTR) is a tetrameric beta-sheet-rich transporter protein directly involved in human amyloid diseases. It was recently found that the isoflavone genistein (GEN) potently inhibits TTR amyloid fibril formation (Green et al., 2005) and is therefore a promising candidate for TTR amyloidosis treatment. Here we used structural and biophysical approaches to characterize genistein binding to the wild type (TTRwt) and to its most frequent amyloidogenic variant, the V30M mutant. In a dose-dependent manner, genistein elicited considerable increases in both mutant and TTRwt stability as demonstrated by high hydrostatic pressure (HHP) and acid-mediated dissociation/denaturation assays. TTR:GEN crystal complexes and isothermal titration calorimetry (ITC) experiments showed that the binding mechanisms of genistein to the TTRwt and to V30M are different and are dependent on apoTTR structure conformations. Furthermore, we could also identify potential allosteric movements caused by genistein binding to the wild type TTR that explains, at least in part, the frequently observed negatively cooperative process between the two sites of TTRwt when binding ligands. These findings show that TTR mutants may present different ligand recognition and therefore are of value in ligand design for inhibiting TTR amyloidosis.


Assuntos
Amiloide/química , Amiloide/metabolismo , Genisteína/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Pré-Albumina/química , Pré-Albumina/metabolismo , Sítio Alostérico , Substituição de Aminoácidos , Amiloide/genética , Amiloidose/etiologia , Amiloidose/metabolismo , Cristalografia por Raios X , Humanos , Concentração de Íons de Hidrogênio , Pressão Hidrostática , Técnicas In Vitro , Ligantes , Modelos Moleculares , Proteínas Mutantes/genética , Pré-Albumina/genética , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Termodinâmica
20.
FEBS Lett ; 584(8): 1609-14, 2010 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-20303962

RESUMO

Coq10p is a protein required for coenzyme Q function, but its specific role is still unknown. It is a member of the START domain superfamily that contains a hydrophobic tunnel implicated in the binding of lipophilic molecules. We used site-directed mutagenesis, statistical coupling analysis and molecular modeling to probe structural determinants in the Coq10p putative tunnel. Four point mutations were generated (coq10-K50E, coq10-L96S, coq10-E105K and coq10-K162D) and their biochemical properties analysed, as well as structural consequences. Our results show that all mutations impaired Coq10p function and together with molecular modeling indicate an important role for the Coq10p putative tunnel.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ubiquinona/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Respiração Celular , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Mutação , Conformação Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA