A carregar...
Projeto de investigação
Sem título
Financiador
Autores
Publicações
Assignment of new roles for malectin-like domains to understand their divergent evolution
Publication . Lourenço, Frederico Miguel da Conceição; Pinheiro, Benedita
Malectin is a highly-conserved animal lectin from the endoplasmic reticulum (ER), with a quality control function in the N-Glycosylation process. It has a β-sandwich core with long loops connecting the β-sheets. Malectin binding-pocket is in the loops region. Several carbohydrate-binding modules (CBMs) discovered in other domains of life that shared sequence homology with the malectin, were classified and grouped as a novel CBM57 family by Carbohydrate-Active Enzymes (CAZy) database. The members of this family are expected to have a highly conserved β-sandwich core, but high variance in the binding-pocket residues.
To investigate if the specificity of these modules is the same as the malectin, a bioinformatic analysis was performed with 315 members of the CBM57 family found in CAZy database. Several programs were used to predict the protein architecture and to analyse the conservation of amino acids sequences, especially in the binding-pocket. Based on this analysis, we predict animal CBM57 modules to have the same specificity as malectin. However, bacterial CBM57 modules in bacteria domain are predicted, after highlighting the modules associated with glycoside hydrolases from family 2, to have various specificities, and thus different biological functions. For verifying these assumptions, a total of 7 CBMs (family 57 and homologous) associated with glycoside hydrolases from family 2 and belonging to the human gut microbiome – Bacteroides ovatus and Bacteroides thetaiotaomicron- were chosen for characterization studies.
A re-cloning was initially performed for the recombinant DNAs, changing the His-tag position. Afterwards, expression tests were realized, in which 2 CBMs of different bacteria were expressed in soluble form. The production of the proteins was then performed at a larger scale, followed by affinity chromatography purification. By the analysis of the gels, the eluted samples had high purity and were suitable for characterization studies.
Glycan microarrays were performed for determining the binding-specificities of the 2 CBM modules. The CBM module from B.thetaiotaomicron revealed high specificity for pectin polysaccharides, possible recognizing α 1-3 linked galacturonic acid and ramnose. For structural characterization by X-ray crystallography, several crystallization trials were performed. Crystals were obtained for the B.thetaiotaomicron CBM module, which diffracted to high resolution. The structure is, yet, to be solved.
The effect of dicarboxymethyl cellulose on the prevention of protein haze formation on white wine
Publication . Gago, Diana; Chagas, Ricardo; Ferreira, Luísa M.; DQ - Departamento de Química; LAQV@REQUIMTE; MDPI - Multidisciplinary Digital Publishing Institute
Wine clarity is a critical aspect in the commercialization of white wines. The formation of wine haze can be attributed to the aggregation and precipitation of heat-unstable wine proteins. Bentonite fining is the commonly used method in winemaking for protein removal, but it is responsible for loss of wine volume and quality. Dicarboxymethyl cellulose (DCMC) was developed as a potential alternative to bentonite. Water-insoluble DCMC was prepared via catalyzed heterogeneous etherification using sodium chloromalonate and potassium iodide. White wine fining trials were benchmarked with different dosages of DCMC against a bentonite. A high-performance liquid chromatography method was optimized for protein quantification. The samples underwent heat stability tests to evaluate wine turbidity before and after fining. Results show that DCMC successfully reduced the wine protein content and turbidity. DCMC produced heat-stable wines with dosages higher than 0.25 g/L. The innovative application of DCMC in the wine sector shows potential due to its ability to stabilize white wines while overcoming problems associated with bentonite, such as lees production and loss of wine, contributing to a more sustainable process.
Structure-function studies can improve binding affinity of cohesin-dockerin interactions for multi-protein assemblies
Publication . Duarte, Marlene; Alves, Victor D.; Correia, Márcia; Caseiro, Catarina; Ferreira, Luís M. A.; Romão, Maria João; Carvalho, Ana Luísa; Najmudin, Shabir; Bayer, Edward A.; Fontes, Carlos M. G. A.; Bule, Pedro; UCIBIO - Applied Molecular Biosciences Unit; DQ - Departamento de Química; Elsevier
The cellulosome is an elaborate multi-enzyme structure secreted by many anaerobic microorganisms for the efficient degradation of lignocellulosic substrates. It is composed of multiple catalytic and non-catalytic components that are assembled through high-affinity protein-protein interactions between the enzyme-borne dockerin (Doc) modules and the repeated cohesin (Coh) modules present in primary scaffoldins. In some cellulosomes, primary scaffoldins can interact with adaptor and cell-anchoring scaffoldins to create structures of increasing complexity. The cellulosomal system of the ruminal bacterium, Ruminococcus flavefaciens, is one of the most intricate described to date. An unprecedent number of different Doc specificities results in an elaborate architecture, assembled exclusively through single-binding-mode type-III Coh-Doc interactions. However, a set of type-III Docs exhibits certain features associated with the classic dual-binding mode Coh-Doc interaction. Here, the structure of the adaptor scaffoldin-borne ScaH Doc in complex with the Coh from anchoring scaffoldin ScaE is described. This complex, unlike previously described type-III interactions in R. flavefaciens, was found to interact in a dual-binding mode. The key residues determining Coh recognition were also identified. This information was used to perform structure-informed protein engineering to change the electrostatic profile of the binding surface and to improve the affinity between the two modules. The results show that the nature of the residues in the ligand-binding surface plays a major role in Coh recognition and that Coh-Doc affinity can be manipulated through rational design, a key feature for the creation of designer cellulosomes or other affinity-based technologies using tailored Coh-Doc interactions.
Tunable Gas Sensing Gels by Cooperative Assembly
Publication . Hussain, Abid; Semeano, Ana T. S.; Palma, Susana I. C. J.; Pina, Ana S.; Almeida, José; Medrado, Bárbara F.; Pádua, Ana C. C. S.; Carvalho, Ana L.; Dionísio, Madalena; Li, Rosamaria W. C.; Gamboa, Hugo; Ulijn, Rein V.; Gruber, Jonas; Roque, Ana C. A.; DQ - Departamento de Química; UCIBIO - Applied Molecular Biosciences Unit; LAQV@REQUIMTE; DF – Departamento de Física; LIBPhys-UNL; WILEY-V C H VERLAG GMBH
The cooperative assembly of biopolymers and small molecules can yield functional materials with precisely tunable properties. Here, the fabrication, characterization, and use of multicomponent hybrid gels as selective gas sensors are reported. The gels are composed of liquid crystal droplets self-assembled in the presence of ionic liquids, which further coassemble with biopolymers to form stable matrices. Each individual component can be varied and acts cooperatively to tune gels' structure and function. The unique molecular environment in hybrid gels is explored for supramolecular recognition of volatile compounds. Gels with distinct compositions are used as optical and electrical gas sensors, yielding a combinatorial response conceptually mimicking olfactory biological systems, and tested to distinguish volatile organic compounds and to quantify ethanol in automotive fuel. The gel response is rapid, reversible, and reproducible. These robust, versatile, modular, pliant electro-optical soft materials possess new possibilities in sensing triggered by chemical and physical stimuli.
Synthetic approaches to a challenging and unusual structure—an amino-pyrrolidine guanine core
Publication . Rippel, Rafael; Pinheiro, Luís; Lopes, Mónica; Lourenço, Ana; Ferreira, Luísa M.; Branco, Paula S.; LAQV@REQUIMTE; MDPI - Multidisciplinary Digital Publishing Institute
The synthesis of an unreported 2-aminopyrrolidine-1-carboxamidine unit is here described for the first time. This unusual and promising structure was attained through the oxidative decarboxylation of amino acids using the pair of reagents, silver(I)/peroxydisulfate (Ag(I)/S2O82−) followed by intermolecular (in the case of L-proline derivative) and intramolecular trapping (in the case of acyl L-arginine) by N-nucleophiles. The L-proline approach has a broader scope for the synthesis of 2-aminopyrrolidine-1-carboxamidine derivatives, whereas the intramolecular cyclization afforded by the L-acylarginines, when applied, results in higher yields. The former allowed the first synthesis of cernumidine, a natural alkaloid isolated in 2011 from Solanum cernuum Vell, as its racemic form.
Unidades organizacionais
Descrição
Palavras-chave
Contribuidores
Financiadores
Entidade financiadora
Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
RECI/BBB-BEP/0124/2012