@article {16921, title = {Tripartite binding mode of cohesin-dockerin complexes from Ruminococcus flavefaciens involving naturally truncated dockerins}, volume = {301}, year = {2025}, note = {n/a}, month = {2025}, pages = {110325}, abstract = {

Polysaccharides in plant cell walls serve as a rich carbon and energy source, yet their structural complexity presents a barrier to efficient degradation. To address this, anaerobic microorganisms like R. flavefaciens have developed sophisticated multi-enzyme complexes known as cellulosomes, which enable the efficient breakdown of these recalcitrant polysaccharides. These complexes are assembled through high-affinity interactions between cohesin (Coh) modules in scaffoldin proteins and dockerin (Doc) modules in cellulosomal enzymes. R. flavefaciens FD-1 harbors one of the most intricate cellulosomes described to date, comprising over 200 Doc-containing proteins encoded in its genome. Despite substantial research on this cellulosome, the role of a group of truncated but functional dockerins, known as group-2 Docs, remains unclear. In this study, we present a detailed structural and binding analysis of a Coh-Doc complex involving the cohesin from the cell-anchoring scaffoldin ScaE and a group-2 Doc that bears only one of the two Ca+2-coordinating loops that characterise the canonical Docs. Our findings reveal a novel tripartite binding mechanism, in which the cohesin can simultaneously bind two distinct dockerin units in three alternative conformations. This discovery provides new insights into the modular versatility of the R. flavefaciens cellulosome and sheds light on the mechanisms that enhance its efficiency in polysaccharide degradation.

}, keywords = {Carbohydrates, cellulosome, cohesin, dockerin, protein assembly, protein complex, protein structure}, isbn = {0021-9258}, url = {https://www.sciencedirect.com/science/article/pii/S0021925825021751}, author = {Marlene Duarte and Carvalho, Ana Lu{\'\i}sa and Ferreira, Magda C. and Caires, Beatriz and Rom{\~a}o, Maria Jo{\~a}o and Prates, Jos{\'e} A. M. and Najmudin, Shabir and Bayer, Edward A. and Fontes, Carlos MGA. and Bule, Pedro} } @inbook {16769, title = {CBMcarb-DB: interface of the three-dimensional landscape of carbohydrate-binding modules}, booktitle = {Carbohydrate Chemistry: Chemical and Biological Approaches Volume 46}, volume = {46}, year = {2024}, note = {n/a}, month = {2024/06/26}, publisher = {Royal Society of Chemistry}, organization = {Royal Society of Chemistry}, abstract = {

Carbohydrate-binding-modules (CBMs) are discrete auxiliary protein modules with a non-catalytic carbohydrate-binding function and that exhibit a great diversity of binding specificities. CBMcarb-DB is a curated database that classifies the three-dimensional structures of CBM{\textendash}carbohydrate complexes determined by single-crystal X-ray diffraction methods and solution NMR spectroscopy. We designed the database architecture and the navigation tools to query the database with the Protein Data Bank (PDB), UniProtKB, and GlyTouCan (universal glycan repository) identifiers. Special attention was devoted to describing the bound glycans using simple graphical representation and numerical format for cross-referencing to other glycosciences and functional data databases. CBMcarb-DB provides detailed information on CBMs and their bound oligosaccharides and features their interactions using several open-access applications. We also describe how the curated information provided by CBMcarb-DB can be integrated with AI algorithms of 3D structure prediction, facilitating structure{\textendash}function studies. Also in this chapter, we discuss the exciting convergence of CBMcarb-DB with the glycan array repository, which serves as a valuable resource for investigating the specific binding interactions between glycans and various biomolecular targets. The interaction of the two fields represents a significant milestone in glycosciences. CBMcarb-DB is freely available at https://cbmdb.glycopedia.eu/ and https://cbmcarb.webhost.fct.unl.pt.

}, isbn = {978-1-83767-217-2}, url = {https://doi.org/10.1039/BK9781837672844-00001}, author = {Ribeiro, D. O. and Bonnardel, F. and Palma, A. S. and Carvalho, A. L. M. and Perez, S.}, editor = {Pilar Rauter, Am{\'e}lia and Queneau, Yves and Palma, Angelina S{\'a}} } @article {16794, title = {Family GH157 enzyme exhibits broad linkage tolerance and a dual endo/exo- β -glucanase activity on β-glucans}, year = {2024}, note = {n/a}, month = {2024}, pages = {137402}, abstract = {

The structural and chemical diversity of β-glucans is reflected on the variety of essential biological roles tackled by these polysaccharides. This natural heterogeneity requires an elaborate assortment of enzymatic mechanisms to assemble, degrade or modify, as well as to extract their full biotechnological potential. Recent metagenomic efforts have provided an unprecedented growth in potential new biocatalysts, most of which remain unconfirmed or uncharacterized. Here we report the first biochemical and structural characterization of two bacterial β-glucanases from the recently created glycoside hydrolase family 157 (LaGH157 and BcGH157) and investigate their molecular basis for substrate hydrolysis. Structural analysis by X-ray crystallography revealed that GH157 enzymes belong to clan GH-A, possessing a (β/α)8-barrel fold catalytic domain, two β-sandwich accessory domains and two conserved catalytic glutamates residues, with relative positions compatible with a retaining mechanism of hydrolysis. Specificity screening and enzyme kinetics suggest that the enzymes prefer mixed-linkage glucans over β-1,3-glucans. Activity screening showed that both enzymes exhibit pH optimum at 6.5 and temperature optimum for LaGH157 and BcGH157 at 25 {\textdegree}C and 48 {\textdegree}C, respectively. Product analysis with HPAEC-PAD and LC-MS revealed that both enzymes are endo-1,3(4)-β-glucanases, capable of cleaving β-1,3 and β-1,4-linked glucoses, when preceded by a β-1,3 linkage. Moreover, BcGH157 needs a minimum of 4 subsites occupied for hydrolysis to occur, while LaGH157 only requires 3 subsites. Additionally, LaGH157 possesses exohydrolytic activity on β-1,3 and branching β-1,6 linkages. This unusual bifunctional endo-1,3(4)/exo-1,3{\textendash}1,6 activity constitutes an expansion on our understanding of β-glucan deconstruction, with the potential to inspire future applications.

}, keywords = {CAZYme, Endo-1,3(4)-β-glucanase, GH157, Glycoside hydrolase, β-Glucans}, isbn = {0141-8130}, url = {https://www.sciencedirect.com/science/article/pii/S0141813024082114}, author = {Caseiro, Catarina and McGregor, Nicholas G.S. and Alves, Victor Diogo and Carvalho, Ana Lu{\'\i}sa and Rom{\~a}o, Maria Jo{\~a}o and Davies, Gideon J. and Fontes, Carlos M. G. A. and Bule, Pedro} } @article {16561, title = {Elucidating the concentration-dependent effects of thiocyanate binding to carbonic anhydrase}, volume = {244}, year = {2023}, note = {n/a}, month = {2023}, pages = {112222}, abstract = {

Many proteins naturally carry metal centers, with a large share of them being in the active sites of several enzymes. Paramagnetic effects are a powerful source of structural information and, therefore, if the native metal is paramagnetic, or it can be functionally substituted with a paramagnetic one, paramagnetic effects can be used to study the metal sites, as well as the overall structure of the protein. One notable example is cobalt(II) substitution for zinc(II) in carbonic anhydrase. In this manuscript we investigate the effects of sodium thiocyanate on the chemical environment of the metal ion of the human carbonic anhydrase II. The electron paramagnetic resonance (EPR) titration of the cobalt(II) protein with thiocyanate shows that the EPR spectrum changes from A-type to C-type on passing from 1:1 to 1:1000-fold ligand excess. This indicates the occurrence of a change in the electronic structure, which may reflect a sizable change in the metal coordination environment in turn caused by a modification of the frozen solvent glass. However, paramagnetic nuclear magnetic resonance (NMR) data indicate that the metal coordination cage remains unperturbed even in 1:1000-fold ligand excess. This result proves that the C-type EPR spectrum observed at large ligand concentration should be ascribed to the low temperature at which EPR measurements are performed, which impacts on the structure of the protein when it is destabilized by a high concentration of a chaotropic agent.

}, keywords = {EPR, Human carbonic anhydrase II, NMR, Paramagnetism, Sodium thiocyanate, Structural biology}, isbn = {0162-0134}, url = {https://www.sciencedirect.com/science/article/pii/S0162013423001046}, author = {Silva, Jos{\'e} Malanho and Cerofolini, Linda and Carvalho, Ana Lu{\'\i}sa and Ravera, Enrico and Fragai, Marco and Parigi, Giacomo and Macedo, Anjos L and Geraldes, Carlos F.G.C. and Luchinat, Claudio} } @article {16257, title = {The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome}, year = {2023}, note = {

n/a

}, month = {2023}, pages = {100084}, abstract = {

TheBacteroides thetaiotaomicronhas developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RGII depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of themoduleat the C-terminal domain, which we designated BT0996C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical β-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.

}, keywords = {Carbohydrate Binding Module, Carbohydrates, Human Gut Microbiota, Rhamnogalacturonan II}, isbn = {2590-1524}, url = {https://www.sciencedirect.com/science/article/pii/S2590152422000253}, author = {Trov{\~a}o, Filipa and Correia, Viviana G. and Louren{\c c}o, Frederico M. and Ribeiro, Diana O. and Carvalho, Ana Lu{\'\i}sa and Palma, Angelina S. and Pinheiro, Benedita A.} } @article {10.3389/fchem.2023.1267563, title = {Hierarchical self-assembly of a reflectin-derived peptide}, journal = {Frontiers in Chemistry}, volume = {11}, year = {2023}, note = {n/a}, abstract = {

Reflectins are a family of intrinsically disordered proteins involved in cephalopod camouflage, making them an interesting source for bioinspired optical materials. Understanding reflectin assembly into higher-order structures by standard biophysical methods enables the rational design of new materials, but it is difficult due to their low solubility. To address this challenge, we aim to understand the molecular self-assembly mechanism of reflectin{\textquoteright}s basic unit{\textemdash}the protopeptide sequence YMDMSGYQ{\textemdash}as a means to understand reflectin{\textquoteright}s assembly phenomena. Protopeptide self-assembly was triggered by different environmental cues, yielding supramolecular hydrogels, and characterized by experimental and theoretical methods. Protopeptide films were also prepared to assess optical properties. Our results support the hypothesis for the protopeptide aggregation model at an atomistic level, led by hydrophilic and hydrophobic interactions mediated by tyrosine residues. Protopeptide-derived films were optically active, presenting diffuse reflectance in the visible region of the light spectrum. Hence, these results contribute to a better understanding of the protopeptide structural assembly, crucial for the design of peptide- and reflectin-based functional materials.

}, issn = {2296-2646}, doi = {10.3389/fchem.2023.1267563}, url = {https://www.frontiersin.org/articles/10.3389/fchem.2023.1267563}, author = {Dias, Ana Margarida Gon{\c c}alves Carvalho and Moreira, In{\^e}s Pimentel and Lychko, Iana and Lopes Soares, C{\'a}tia and Nurrito, Arianna and Moura Barbosa, Arm{\'e}nio Jorge and Lutz-Bueno, Viviane and Mezzenga, Raffaele and Carvalho, Ana Lu{\'\i}sa and Pina, Ana Sofia and Roque, Ana Cec{\'\i}lia Afonso} } @article {ijms24076246, title = {Structural Characterization of Neisseria gonorrhoeae Bacterial Peroxidase\—Insights into the Catalytic Cycle of Bacterial Peroxidases}, journal = {International Journal of Molecular Sciences}, volume = {24}, number = {7}, year = {2023}, note = {n/a}, abstract = {

Neisseria gonorrhoeae is an obligate human pathogenic bacterium responsible for gonorrhea, a sexually transmitted disease. The bacterial peroxidase, an enzyme present in the periplasm of this bacterium, detoxifies the cells against hydrogen peroxide and constitutes one of the primary defenses against exogenous and endogenous oxidative stress in this organism. The 38 kDa heterologously produced bacterial peroxidase was crystallized in the mixed-valence state, the active state, at pH 6.0, and the crystals were soaked with azide, producing the first azide-inhibited structure of this family of enzymes. The enzyme binds exogenous ligands such as cyanide and azide, which also inhibit the catalytic activity by coordinating the P heme iron, the active site, and competing with its substrate, hydrogen peroxide. The inhibition constants were estimated to be 0.4 \± 0.1 \µM and 41 \± 5 mM for cyanide and azide, respectively. Imidazole also binds and inhibits the enzyme in a more complex mechanism by binding to P and E hemes, which changes the reduction potential of the latest heme. Based on the structures now reported, the catalytic cycle of bacterial peroxidases is revisited. The inhibition studies and the crystal structure of the inhibited enzyme comprise the first platform to search and develop inhibitors that target this enzyme as a possible new strategy against N. gonorrhoeae.

}, issn = {1422-0067}, doi = {10.3390/ijms24076246}, url = {https://www.mdpi.com/1422-0067/24/7/6246}, author = {N{\'o}brega, Cl{\'a}udia S. and Carvalho, Ana Lu{\'\i}sa and Rom{\~a}o, Maria Jo{\~a}o and Pauleta, Sofia R} } @article {DUARTE202355, title = {Structure-function studies can improve binding affinity of cohesin-dockerin interactions for multi-protein assemblies}, journal = {International Journal of Biological Macromolecules}, volume = {224}, year = {2023}, note = {n/a}, pages = {55-67}, abstract = {

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.

}, keywords = {Biomass degradation, Carbohydrates, cellulosome, cohesin, dockerin, protein complex}, issn = {0141-8130}, doi = {https://doi.org/10.1016/j.ijbiomac.2022.10.102}, url = {https://www.sciencedirect.com/science/article/pii/S0141813022023480}, author = {Marlene Duarte and Alves, Victor D. and Correia, M{\'a}rcia and Caseiro, Catarina and Ferreira, Lu{\'\i}s M A and Rom{\~a}o, Maria Jo{\~a}o and Carvalho, Ana Lu{\'\i}sa and Najmudin, Shabir and Bayer, Edward A. and Fontes, Carlos M. G. A. and Bule, Pedro} } @article {MOREIRA2022100290, title = {Synergy between silk fibroin and ionic liquids for active gas-sensing materials}, journal = {Materials Today Bio}, year = {2022}, note = {n/a}, pages = {100290}, abstract = {

Silk fibroin is a biobased material with excellent biocompatibility and mechanical properties, but its use in bioelectronics is hampered by the difficult dissolution and low intrinsic conductivity. Some ionic liquids are known to dissolve fibroin but removed after fibroin processing. However, ionic liquids and fibroin can cooperatively give rise to functional materials, and there are untapped opportunities in this combination. The dissolution of fibroin, followed by gelation, in designer ionic liquids from the imidazolium chloride family with varied alkyl chain lengths (2{\textendash}10 carbons) is shown here. The alkyl chain length of the anion has a large impact on fibroin secondary structure which adopts unconventional arrangements, yielding robust gels with distinct hierarchical organization. Furthermore, and due to their remarkable air-stability and ionic conductivity, fibroin ionogels are exploited as active electrical gas sensors in an electronic nose revealing the unravelled possibilities of fibroin in soft and flexible electronics.

}, keywords = {Bioelectronics, gas sensing, Ionic conductivity, ionic liquids, Physical ionogels, Silk fibroin}, issn = {2590-0064}, doi = {https://doi.org/10.1016/j.mtbio.2022.100290}, url = {https://www.sciencedirect.com/science/article/pii/S2590006422000886}, author = {In{\^e}s P. Moreira and Esteves, Carina and Palma, Susana I.C.J. and Ramou, Efthymia and Ana L.M. Carvalho and Roque, Ana C. A.} } @article {Esteves2022, title = {{Tackling Humidity with Designer Ionic Liquid-Based Gas Sensing Soft Materials}}, journal = {Advanced Materials}, year = {2022}, note = {n/a}, abstract = {

Relative humidity is simultaneously a sensing target and a contaminant in gas and volatile organic compound (VOC) sensing systems, where strategies to control humidity interference are required. An unmet challenge is the creation of gas-sensitive materials where the response to humidity is controlled by the material itself. Here, humidity effects are controlled through the design of gelatin formulations in ionic liquids without and with liquid crystals as electrical and optical sensors, respectively. In this design, the anions [DCA]- and [Cl]- of room temperature ionic liquids from the 1-butyl-3-methylimidazolium family tailor the response to humidity and, subsequently, sensing of VOCs in dry and humid conditions. Due to the combined effect of the materials formulations and sensing mechanisms, changing the anion from [DCA]- to the much more hygroscopic [Cl]-, leads to stronger electrical responses and much weaker optical responses to humidity. Thus, either humidity sensors or humidity-tolerant VOC sensors that do not require sample preconditioning or signal processing to correct humidity impact are obtained. With the wide spread of 3D- and 4D-printing and intelligent devices, the monitoring and tuning of humidity in sustainable biobased materials offers excellent opportunities in e-nose sensing arrays and wearable devices compatible with operation at room conditions.

}, issn = {15214095}, doi = {10.1002/adma.202107205}, author = {Esteves, Carina and Palma, Susana I.C.J. and Costa, Henrique M.A. and Alves, Cl{\'a}udia and Santos, Gon{\c c}alo M.C. and Ramou, Efthymia and Carvalho, Ana Lu{\'\i}sa and Alves, Vitor and Roque, Ana C. A.} } @article {15279, title = {Structural insights into the molecular recognition mechanism of the cancer and pathogenic epitope, LacdiNAc by immune-related lectins}, journal = {Chemistry {\textendash} A European JournalChemistry {\textendash} A European Journal}, volume = {n/a}, year = {2021}, note = {

https://doi.org/10.1002/chem.202100800

}, month = {2021}, publisher = {John Wiley \& Sons, Ltd}, abstract = {

Interactions of glycan-specific epitopes to human lectin receptors represent novel immune checkpoints for investigating cancer and infection diseases. By employing a multidisciplinary approach that combines isothermal titration calorimetry, NMR spectroscopy, molecular dynamics simulations, and X-ray crystallography, we disclosed the molecular determinants that govern the recognition of the tumour and pathogenic glycobiomarker LacdiNAc (GalNAc?1-4GlcNAc, LDN), including their comparison with the ubiquitous LacNAc epitope (Gal?1-4GlcNAc, LN), by two human immune-related lectins, galectin-3 (hGal-3) and the macrophage galactose C-type lectin (hMGL). A different mechanism of binding and interactions is observed for the hGal-3/LDN and hMGL/LDN complexes, which explains the remarkable difference in the binding specificity of LDN and LN by these two lectins. The new structural clues reported herein are fundamental for the chemical design of mimetics targeting hGal-3/hMGL recognition process.

}, keywords = {glycan-protein interactions, hGal-3, hMGL, LacdiNAc, molecular recognition}, isbn = {0947-6539}, url = {https://doi.org/10.1002/chem.202100800}, author = {Lima, Carlos D. L. and Helena Coelho and Gimeno, Ana and Trov{\~a}o, Filipa and Diniz, Ana and Dias, Jorge S. and Jes{\'u}s Jim{\'e}nez-Barbero and Corzana, Francisco and Carvalho, Ana Lu{\'\i}sa and Cabrita, Eurico J. and Marcelo, Filipa} } @article {15549, title = {Mapping Molecular Recognition of β1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont Bacteroides ovatus}, journal = {Microbiology SpectrumMicrobiology Spectrum}, year = {2021}, note = {

doi: 10.1128/Spectrum.01826-21

}, pages = {e01826-21}, publisher = {American Society for Microbiology}, abstract = {

n/a

}, url = {https://doi.org/10.1128/Spectrum.01826-21}, author = {Correia Viviana G. and Trov{\~a}o Filipa and Pinheiro Benedita A. and Br{\'a}s Joana L. A. and Silva Lisete M. and Nunes Cl{\'a}udia and Coimbra Manuel A. and Liu Yan and Feizi Ten and Fontes Carlos M. G. A. and Mulloy Barbara and Chai Wengang and Carvalho Ana Lu{\'\i}sa and Palma Angelina S.} } @article {DUARTE2021100552, title = {A dual cohesin{\textendash}dockerin complex binding mode in Bacteroides cellulosolvens contributes to the size and complexity of its cellulosome}, journal = {Journal of Biological Chemistry}, volume = {296}, year = {2021}, note = {n/a}, pages = {100552}, abstract = {

The Cellulosome is an intricate macromolecular protein complex that centralizes the cellulolytic efforts of many anaerobic microorganisms through the promotion of enzyme synergy and protein stability. The assembly of numerous carbohydrate processing enzymes into a macromolecular multiprotein structure results from the interaction of enzyme-borne dockerin modules with repeated cohesin modules present in noncatalytic scaffold proteins, termed scaffoldins. Cohesin{\textendash}dockerin (Coh-Doc) modules are typically classified into different types, depending on structural conformation and cellulosome role. Thus, type I Coh-Doc complexes are usually responsible for enzyme integration into the cellulosome, while type II Coh-Doc complexes tether the cellulosome to the bacterial wall. In contrast to other known cellulosomes, cohesin types from Bacteroides cellulosolvens, a cellulosome-producing bacterium capable of utilizing cellulose and cellobiose as carbon sources, are reversed for all scaffoldins, i.e., the type II cohesins are located on the enzyme-integrating primary scaffoldin, whereas the type I cohesins are located on the anchoring scaffoldins. It has been previously shown that type I B.~cellulosolvens interactions possess a dual-binding mode that adds flexibility to scaffoldin assembly. Herein, we report the structural mechanism of enzyme recruitment into B.~cellulosolvens cellulosome and the identification of the molecular determinants of its type II cohesin{\textendash}dockerin interactions. The results indicate that, unlike other type II complexes, these possess a dual-binding mode of interaction, akin to type I complexes. Therefore, the plasticity of dual-binding mode interactions seems to play a pivotal role in the assembly of B.~cellulosolvens cellulosome, which is consistent with its unmatched complexity and size.

}, keywords = {cellulase, cellulose, cellulosome, cohesin, crystal structure, dockerin, dual-binding, protein complex}, issn = {0021-9258}, doi = {https://doi.org/10.1016/j.jbc.2021.100552}, url = {https://www.sciencedirect.com/science/article/pii/S0021925821003306}, author = {Marlene Duarte and Viegas, Aldino and Alves, Victor D. and Prates, Jos{\'e} A. M. and Ferreira, Lu{\'\i}s M A and Najmudin, Shabir and Cabrita, Eurico J. and Carvalho, Ana Lu{\'\i}sa and Fontes, Carlos M. G. A. and Bule, Pedro} } @article {D0CE01529F, title = {Magnetic particles used in a new approach for designed protein crystallization}, journal = {CrystEngComm}, volume = {23}, year = {2021}, note = {n/a}, pages = {1083-1090}, publisher = {The Royal Society of Chemistry}, abstract = {

After more than one hundred and thirty thousand protein structures determined by X-ray crystallography{,} the challenge of protein crystallization for 3D structure determination remains. In the quest for additives for efficient protein crystallization{,} inorganic materials emerge as an alternative. Magnetic particles (MPs) are versatile inorganic materials{,} easy to use{,} modify and manipulate in a wide range of biological assays. The potential of using functionalised MPs as crystallization chaperones for protein crystallization was shown in this work. MPs with distinct coatings were rationally designed to promote protein crystallization by affinity-triggered heterogeneous nucleation. Hen egg white lysozyme (HEWL) and trypsin{,} were crystallized in the presence of MPs either bare or coated with a polysaccharide (chitin) or a protein (casein){,} respectively. The addition of MPs was characterized in terms of bound protein to the MPs{,} crystal morphology{,} time-lapse of crystal emergence{,} crystallization yield fold change and crystal diffraction quality for structure determination. The MPs additives have shown to bind to the respective target protein{,} and to promote nucleation and crystal growth without compromising crystal morphology. On the other hand{,} MPs addition led to faster detectable crystal emergence and up to 13 times higher crystallization yield{,} addressing some the challenges in protein crystallization{,} the main bottleneck of macromolecular crystallography. Structure determination of the protein crystallized in the presence of MPs revealed that the structural characteristics of the protein remained unchanged{,} as shown by the superposition with PDB annotated proteins. Moreover{,} and unlike most reported cases{,} it was possible to exclude the inhibitor benzamidine during trypsin crystallisation{,} which is a remarkable result opening new prospects in enzyme engineering and drug design. Our results show that MPs coated with affinity ligands to target proteins can be used as controlled and tailor-made crystallization inducers.

}, doi = {10.1039/D0CE01529F}, url = {http://dx.doi.org/10.1039/D0CE01529F}, author = {Raquel dos Santos and Rom{\~a}o, Maria Jo{\~a}o and Ana Cec{\'\i}lia A. Roque and Carvalho, Ana Lu{\'\i}sa} } @article {membranes11080549, title = {Protein Crystallization in a Microfluidic Contactor with Nafion{\textregistered}117 Membranes}, journal = {Membranes}, volume = {11}, number = {8}, year = {2021}, note = {n/a}, abstract = {

Protein crystallization still remains mostly an empirical science, as the production of crystals with the required quality for X-ray analysis is dependent on the intensive screening of the best protein crystallization and crystal{\textquoteright}s derivatization conditions. Herein, this demanding step was addressed by the development of a high-throughput and low-budget microfluidic platform consisting of an ion exchange membrane (117 Nafion{\textregistered} membrane) sandwiched between a channel layer (stripping phase compartment) and a wells layer (feed phase compartment) forming 75 independent micro-contactors. This microfluidic device allows for a simultaneous and independent screening of multiple protein crystallization and crystal derivatization conditions, using Hen Egg White Lysozyme (HEWL) as the model protein and Hg2+ as the derivatizing agent. This microdevice offers well-regulated crystallization and subsequent crystal derivatization processes based on the controlled transport of water and ions provided by the 117 Nafion{\textregistered} membrane. Diffusion coefficients of water and the derivatizing agent (Hg2+) were evaluated, showing the positive influence of the protein drop volume on the number of crystals and crystal size. This microfluidic system allowed for crystals with good structural stability and high X-ray diffraction quality and, thus, it is regarded as an efficient tool that may contribute to the enhancement of the proteins{\textquoteright} crystals structural resolution.

}, issn = {2077-0375}, doi = {10.3390/membranes11080549}, url = {https://www.mdpi.com/2077-0375/11/8/549}, author = {Polino, M. and Rho, H. S. and Pina, M. P. and Mallada, R. and Carvalho, AL and Rom{\~a}o, MJ and Coelhoso, Isabel and Gardeniers, J. G. E. and Crespo, J. G. and Portugal, Carla A. M.} } @article {Goodfellow2021, title = {{The SOUL family of heme-binding proteins: Structure and function 15~years later}}, journal = {Coordination Chemistry Reviews}, volume = {448}, year = {2021}, note = {n/a}, pages = {214189}, abstract = {

The SOUL, or heme-binding protein HBP/SOUL, family represents a group of evolutionary conserved putative heme-binding proteins that contains a number of members in animal, plant andbacterial species. The structures of the murine form of HEBP1, or p22HBP, and the human form of HEBP2, or SOUL, have been determined in 2006 and 2011 respectively. In this work we discuss the structures of HEBP1 and HEBP2 in light of new X-ray data for heme bound murine HEBP1. The interaction between tetrapyrroles and HEBP1, initially proven to be hydrophobic in nature, was thought to also involve electrostatic interactions between heme propionate groups and positively charged amino acid side chains. However, the new X-ray structure, and results from murine HEBP1 variants and human HEBP1, confirm the hydrophobic nature of the heme-HEBP1 interaction, resulting in Kd values in the low nanomolar range, and rules out any electrostatic stabilization. Results from NMR relaxation time measurements for human HEBP1 describe a rigid globular protein with no change in motional regime upon heme binding. X-ray structures deposited in the PDB for human HEBP2 are very similar to each other and to the new heme-bound murine HEBP1 X-ray structure (backbone rmsd ca. 1~{\AA}). Results from a HSQC spectrum centred on the histidine side chain N$δ$-proton region for HEBP2 confirm that HEBP2 does not bind heme via H42 as no chemical shift differences were observed upon heme addition for backbone NH and N$δ$ protons. A survey of the functions attributed to HEBP1 and HEBP2 over the last 20~years span a wide range of cellular pathways. Interestingly, many of them are specific to higher eukaryotes, particularly mammals and a potential link between heme release under oxidative stress and human HEBP1 is also examined using recent data. However, at the present moment, trying to relate function to the involvement of heme or tetrapyrrole binding, specifically, makes little sense with our current biological knowledge and can only be applied to HEBP1, as HEBP2 does not interact with heme. We suggest that it may not be justified to call this very small family of proteins, heme-binding proteins. The family may be more correctly called {\textquotedblleft}the SOUL family of proteins related to cellular fate{\textquotedblright} as, even though only HEBP1 binds heme tightly, both proteins may be involved in cell survival and/or proliferation.

}, keywords = {Function, HEBP1, HEBP2, NMR spectroscopy, SOUL protein, Structure, X-ray crystallography}, issn = {0010-8545}, doi = {https://doi.org/10.1016/j.ccr.2021.214189}, url = {https://www.sciencedirect.com/science/article/pii/S001085452100463X}, author = {Goodfellow, Brian J. and Freire, Filipe and Carvalho, Ana Lu{\'\i}sa and Aveiro, Susana S. and Charbonnier, Peggy and Moulis, Jean-Marc and Delgado, Leonildo and Ferreira, Gloria C and Rodrigues, Jo{\~a}o E and Poussin-Courmontagne, Pierre and Birck, Catherine and McEwen, Alastair and Macedo, Anjos L} } @article {14903, title = {Magnetic Precipitation: A New Platform for Protein Purification}, journal = {Biotechnology JournalBiotechnology Journal}, volume = {n/a}, year = {2020}, note = {

doi: 10.1002/biot.202000151

}, month = {2020}, pages = {2000151}, publisher = {John Wiley \& Sons, Ltd}, abstract = {

One of the trends in downstream processing comprises the use of ?anything-but-chromatography? methods to overcome the current downfalls of standard packed-bed chromatography. Precipitation and magnetic separation are two techniques already proven to accomplish protein purification from complex media, yet never used in synergy. With the aim to capture antibodies directly from crude extracts, a new approach combining precipitation and magnetic separation was developed and named as affinity magnetic precipitation. A precipitation screening, based on the Hofmeister series, and a commercial precipitation kit were tested with affinity magnetic particles to assess the best condition for antibody capture from human serum plasma and clarified cell supernatant. The best conditions were obtained when using PEG3350 as precipitant at 4{\textdegree}C for 1h, reaching 80\% purity and 50\% recovery of polyclonal antibodies from plasma, and 99\% purity with 97\% recovery yield of anti-TNFα mAb from cell supernatants. These results show that the synergetic use of precipitation and magnetic separation can represent an alternative for the efficient capture of antibodies. This article is protected by copyright. All rights reserved

}, keywords = {affinity magnetic precipitation, antibody purification, downstream, magnetic nanoparticles, method development}, isbn = {1860-6768}, url = {https://doi.org/10.1002/biot.202000151}, author = {Raquel dos Santos and Iria, In{\^e}s and Manuel, Ana M and Leandro, Ana P and Madeira, Catarina A C and Goncalves, Joao and Carvalho, Ana Lu{\'\i}sa and Roque, Ana Cec{\'\i}lia} } @article {13516, title = {SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations}, volume = {1864}, year = {2020}, note = {n/a}, month = {2020}, pages = {129440}, abstract = {

BackgroundHalf of human cancers harbour TP53 mutations that render p53 inactive as a tumor suppressor. As such, reactivation of mutant (mut)p53 through restoration of wild-type (wt)-like function represents one of the most promising therapeutic strategies in cancer treatment. Recently, we have reported the (S)-tryptophanol-derived oxazoloisoindolinone SLMP53-1 as a new reactivator of wt and mutp53 R280K with in vitro and in vivo p53-dependent antitumor activity. The present work aimed a mechanistic elucidation of mutp53 reactivation by SLMP53-1.
Methods and results
By cellular thermal shift assay (CETSA), it is shown that SLMP53-1 induces wt and mutp53 R280K thermal stabilization, which is indicative of intermolecular interactions with these proteins. Accordingly, in silico studies of wt and mutp53 R280K DNA-binding domain with SLMP53-1 unveiled that the compound binds at the interface of the p53 homodimer with the DNA minor groove. Additionally, using yeast and p53-null tumor cells ectopically expressing distinct highly prevalent mutp53, the ability of SLMP53-1 to reactivate multiple mutp53 is evidenced.
Conclusions
SLMP53-1 is a p53-activating agent with the ability to directly target wt and a set of hotspot mutp53.
General Significance
This work reinforces the encouraging application of SLMP53-1 in the personalized treatment of cancer patients harboring distinct p53 status.

}, keywords = {Cancer, Chemotherapy, Mutant, p53, Reactivator}, isbn = {0304-4165}, url = {http://www.sciencedirect.com/science/article/pii/S0304416519302260}, author = {Gomes, Ana Sara and Ramos, Helena and Gomes, Sara and Loureiro, Joana B. and Soares, Joana and Barcherini, Valentina and Monti, Paola and Fronza, Gilberto and Oliveira, Carla and Domingues, Luc{\'\i}lia and Bastos, Margarida and Dourado, Daniel F.A.R. and Carvalho, Ana Lu{\'\i}sa and Rom{\~a}o, Maria Jo{\~a}o and Pinheiro, Benedita and Marcelo, Filipa and Carvalho, Alexandra and Santos, Maria M.M. and Saraiva, Luc{\'\i}lia} } @article {https://doi.org/10.1111/febs.15162, title = {Molecular basis for the preferential recognition of β1,3-1,4-glucans by the family 11 carbohydrate-binding module from Clostridium~thermocellum}, journal = {The FEBS Journal}, volume = {287}, number = {13}, year = {2020}, note = {n/a}, pages = {2723-2743}, abstract = {

Understanding the specific molecular interactions between proteins and β1,3-1,4-mixed-linked d-glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate-binding module from Clostridium~thermocellum (CtCBM11) is known for its binding preference for β1,3-1,4-mixed-linked over β1,4-linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to β1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the β1,3-linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site-directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of β1,3-1,4-mixed-linked glucans. This is mediated by a conformation{\textendash}selection mechanism of the ligand in the binding cleft through CH-π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a β1,3-linkage at the reducing end and at specific positions along the β1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked β-glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture. Database Structural data are available in the Protein Data Bank under the accession codes 6R3M and 6R31.

}, keywords = {3-1, 4-mixed-linked glucans, carbohydrate specificity, carbohydrate-binding module, cellulosome, Clostridium~thermocellum, β1}, doi = {https://doi.org/10.1111/febs.15162}, url = {https://febs.onlinelibrary.wiley.com/doi/abs/10.1111/febs.15162}, author = {Ribeiro, Diana O. and Viegas, Aldino and Pires, Virg{\'\i}nia M R and Medeiros-Silva, Jo{\~a}o and Bule, Pedro and Chai, Wengang and Marcelo, Filipa and Fontes, Carlos M. G. A. and Cabrita, Eurico J. and Palma, Angelina S. and Carvalho, Ana Lu{\'\i}sa} } @article {https://doi.org/10.1002/ejic.202000423, title = {Synthesis, Crystal Structure, and DFT Study of Two New Dinuclear Copper(I) Complexes Bearing Ar-BIAN Ligands Functionalized with NO2 Groups}, journal = {European Journal of Inorganic Chemistry}, volume = {2020}, number = {30}, year = {2020}, note = {n/a}, pages = {2900-2911}, abstract = {

{Two new bis(aryl-imino)-acenaphthene, Ar-BIAN (Ar = 2

}, keywords = {Copper(I), crystal structure, Density Function Calculations, Functionalized Ar-BIAN, synthesis}, doi = {https://doi.org/10.1002/ejic.202000423}, url = {https://chemistry-europe.onlinelibrary.wiley.com/doi/abs/10.1002/ejic.202000423}, author = {Outis, Mani and Vitor Rosa, and Laia, C{\'e}sar AT and Lima, Jo{\~a}o Carlos and Barroso, S{\'o}nia and Carvalho, Ana Lu{\'\i}sa and Calhorda, Maria Jos{\'e} and Avil{\'e}s, Teresa} } @article {13104, title = {N-Heterocyclic Olefin Catalysis for the Ring Opening of Cyclic Amidine Compounds: A Pathway to the Synthesis of ε-Caprolactam- and γ-Lactam-Derived Amines}, journal = {The Journal of Organic ChemistryThe Journal of Organic Chemistry}, year = {2019}, note = {

doi: 10.1021/acs.joc.8b02823

}, month = {2019}, publisher = {American Chemical Society}, abstract = {n/a}, isbn = {0022-3263}, url = {https://doi.org/10.1021/acs.joc.8b02823}, author = {Peixoto, Daniela and Malta, Gabriela and Cruz, Hugo and Barroso, S{\'o}nia and Carvalho, Ana Lu{\'\i}sa and Ferreira, Lu{\'\i}sa M. and Paula S. Branco} } @inbook {doi:10.1002/9781119282549.ch1, title = {Resistance to Aminoglycosides}, booktitle = {Antibiotic Drug Resistance}, year = {2019}, note = {

n/a

}, pages = {1-38}, publisher = {John Wiley \& Sons, Ltd}, organization = {John Wiley \& Sons, Ltd}, chapter = {1}, abstract = {

Summary The emergence of bacterial resistance to different antibiotics in clinical use, together with the knowledge on the mechanisms by which bacteria resist the action of aminoglycosides, have contributed to the renewed interest in these molecules as potential antimicrobials. Here, we give an overview on natural and semisynthetic aminoglycosides and their structural features and modes of action, focusing on the structural insight underlying resistance mechanisms. Developments on carbohydrate chemistry and microarray technology are highlighted as powerful approaches toward generation of new aminoglycosides and for screening their interactions with RNAs and proteins. The link between antibiotic uptake and the human gut microbiome is also addressed, focusing on gut microbiome function and composition, antibiotic-induced alterations in host health, and antibiotic resistance. In addition, strategies to modulate human microbiome responses to antibiotics are discussed as novel approaches for aminoglycoside usage and for the effectiveness of antibiotic therapy.

}, keywords = {aminoglycosides, antibiotics, glycomics, gut microbiome, microarrays, modifying enzymes, resistance mechanisms}, isbn = {9781119282549}, doi = {10.1002/9781119282549.ch1}, url = {https://onlinelibrary.wiley.com/doi/abs/10.1002/9781119282549.ch1}, author = {Correia, Viviana G. and Pinheiro, Benedita A. and Carvalho, Ana Lu{\'\i}sa and Palma, Angelina S.} } @inbook {Carvalho2018, title = {{CHAPTER 2 Structural Elucidation of Macromolecules}}, booktitle = {Essential Techniques for Medical and Life Scientists: A Guide to Contemporary Methods and Current Applications with the Protocols}, year = {2018}, note = {

n/a

}, month = {sep}, pages = {30{\textendash}91}, publisher = {BENTHAM SCIENCE PUBLISHERS}, organization = {BENTHAM SCIENCE PUBLISHERS}, abstract = {

n/a

}, keywords = {cryo-em, drug design, ligand interactions, macromolecular structure, NMR, protein-, saxs, X-ray crystallography}, doi = {10.2174/9781681087092118010005}, url = {http://www.eurekaselect.com/node/165742}, author = {Carvalho, Ana Lu{\'\i}sa and Santos-Silva, Teresa and Rom{\~a}o, Maria Jo{\~a}o and Eurico, J and Marcelo, Filipa} } @article {12365, title = {Higher order scaffoldin assembly in Ruminococcus flavefaciens cellulosome is coordinated by a discrete cohesin-dockerin interaction}, journal = {Scientific Reports}, volume = {8}, year = {2018}, note = {

n/a

}, month = {2018}, pages = {6987}, abstract = {

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

}, isbn = {2045-2322}, url = {https://doi.org/10.1038/s41598-018-25171-8}, author = {Bule, Pedro and Pires, Virg{\'\i}nia M R and Alves, Victor D. and Carvalho, Ana Lu{\'\i}sa and Prates, Jos{\'e} A. M. and Ferreira, Lu{\'\i}s M A and Smith, Steven P. and Gilbert, Harry J. and Noach, Ilit and Bayer, Edward A. and Najmudin, Shabir and Fontes, Carlos M. G. A.} } @article {12521, title = {Molecular recognition of a Thomsen-Friedenreich antigen mimetic targeting human galectin-3}, journal = {ChemMedChem}, volume = {Aug 9. doi: 10.1002/cmdc.201800525. [Epub ahead of print]}, year = {2018}, note = {

doi: 10.1002/cmdc.201800525

}, month = {2018}, publisher = {Wiley-Blackwell}, abstract = {

Overexpression of the Thomsen-Friedenreich (TF) antigen in cell membrane proteins occurs in 90\% of adenocarcinomas. Additionally, the binding of the TF-antigen to human galectin-3 (Gal-3), also frequently overexpressed in malignancy, promotes cancer progression and metastasis. In this context, structures that interfere with this specific interaction display the potential to prevent cancer metastasis. Herein, a multidisciplinary approach, combining the optimized synthesis of a TF-antigen mimetic with NMR, X-ray crystallography methods and isothermal titration calorimetry assays has been employed to unravel the molecular structural details that govern the Gal-3/TF-mimetic interaction. The TF-mimetic presents a binding affinity for Gal-3 similar to the TF-natural antigen and retains the binding epitope and the bioactive conformation observed for the native antigen. Furthermore, from a thermodynamic perspective a decrease in the enthalpic contribution was observed for the Gal-3/TF-mimetic complex, however this behaviour is compensated by a favourable entropy gain. From a structural perspective, these results establish our TF-mimetic as a scaffold to design multivalent solutions to potentially interfere with Gal-3 aberrant interactions and likely be used to hamper Gal-3-mediated cancer cells adhesion and metastasis.

}, keywords = {galectin-3, molecular recognition, NMR spectroscopy, Tumour-associated carbohydrate antigens, X-ray crystallography}, isbn = {1860-7179}, url = {https://doi.org/10.1002/cmdc.201800525}, author = {Santarsia, Sabrina and Grosso, Ana Sofia and Trov{\~a}o, Filipa and Jes{\'u}s Jim{\'e}nez-Barbero and Carvalho, Ana Lu{\'\i}sa and Nativi, Cristina and Marcelo, Filipa} } @article {EJOC:EJOC201701500, title = {Synthesis, cytotoxicity evaluation in human cell lines and in vitro DNA interaction of a hetero arylidene-9(10H)-anthrone}, journal = {European Journal of Organic Chemistry}, year = {2018}, note = {

n/a

}, pages = {n/a{\textendash}n/a}, abstract = {

A new and never yet reported hetero arylidene-9(10H)-anthrone structure (4) was unexpectedly isolated on reaction of 1,2-dimethyl-3-ethylimidazolium iodide (2) and 9-anthracenecarboxaldehyde (3) under basic conditions. Its structure was unequivocally attributed by X-ray crystallography. No cytotoxicity in human healthy fibroblasts and in two different cancer cell lines was observed indicating its applicability in biological systems. Compound 4 interacts with CT-DNA by intercalation between the adjacent base pairs of DNA with a high binding affinity (Kb = 2.0({\textpm} 0.20) x 105 M-1) which is 10x higher than that described for doxorubicin (Kb = 3.2 ({\textpm}0.23) {\texttimes} 104 M-1). Furthermore, compound 4 quenches the fluorescence emission of GelRed-CT-DNA system with a quenching constant (KSV) of 3.3({\textpm}0.3) x 103 M-1 calculated by the Stern-Volmer equation.

}, keywords = {anthracenecarboxaldehyde, arylidene anthrone, Decarbonylation, DNA intercalation, Imidazolium salt}, issn = {1099-0690}, doi = {10.1002/ejoc.201701500}, url = {http://dx.doi.org/10.1002/ejoc.201701500}, author = {Paula S. Branco and Peixoto, Daniela and Figueiredo, Margarida and Malta, Gabriela and Roma-Rodrigues, Catarina and Batista, Pedro Viana and Fernandes, Alexandra R. and Barroso, S{\'o}nia and Carvalho, Ana Luisa and Afonso, Carlos A. M. and Ferreira, Lu{\'\i}sa Maria} } @article {ijms19041184, title = {The Crystal Structure of the R280K Mutant of Human p53 Explains the Loss of DNA Binding}, journal = {International Journal of Molecular Sciences}, volume = {19}, number = {4}, ARTICLE NUMBER = {1184}, year = {2018}, note = {n/a}, abstract = {

The p53 tumor suppressor is widely found to be mutated in human cancer. This protein is regarded as a molecular hub regulating different cell responses, namely cell death. Compelling data have demonstrated that the impairment of p53 activity correlates with tumor development and maintenance. For these reasons, the reactivation of p53 function is regarded as a promising strategy to halt cancer. In the present work, the recombinant mutant p53R280K DNA binding domain (DBD) was produced for the first time, and its crystal structure was determined in the absence of DNA to a resolution of 2.0 {\r A}. The solved structure contains four molecules in the asymmetric unit, four zinc(II) ions, and 336 water molecules. The structure was compared with the wild-type p53 DBD structure, isolated and in complex with DNA. These comparisons contributed to a deeper understanding of the mutant p53R280K structure, as well as the loss of DNA binding related to halted transcriptional activity. The structural information derived may also contribute to the rational design of mutant p53 reactivating molecules with potential application in cancer treatment.

}, issn = {1422-0067}, doi = {10.3390/ijms19041184}, url = {http://www.mdpi.com/1422-0067/19/4/1184}, author = {Gomes, Ana Sara and Trov{\~a}o, Filipa and Andrade Pinheiro, Benedita and Freire, Filipe and Gomes, Sara and Oliveira, Carla and Domingues, Luc{\'\i}lia and Rom{\~a}o, Maria Jo{\~a}o and Saraiva, Luc{\'\i}lia and Carvalho, Ana Lu{\'\i}sa} } @article {Kumar2018, title = {Novel insights into the degradation of β-1,3-glucans by the cellulosome of Clostridium thermocellum revealed by structure and function studies of a family 81 glycoside hydrolase}, journal = {International Journal of Biological Macromolecules}, year = {2018}, note = {n/a}, pages = {-}, abstract = {

Abstract The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a β-1,3-glucanase belonging to cellulosomal complex. The gene encoding \{GH81\} from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of ~82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 {\textdegree}C. CtLam81A displayed Km, 2.1 {\textpm} 0.12 mg/ml and Vmax, 109 {\textpm} 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 {\textdegree}C to 96 {\textdegree}C by Ca2+ or Mg2+ ions and decreased to 82 {\textdegree}C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. \{TLC\} and MALDI-TOF analysis of β-1,3-glucan hydrolysed products released initially, showed β-1,3-glucan-oligosaccharides degree of polymerization (DP) from \{DP2\} to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 {\r A} resolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 β-strands forming a four-domain structure viz. a β-sandwich domain I at N-terminal, an α/β-domain II, an (α/α)6 barrel domain III, and a small 5-stranded β-sandwich domain IV.

}, keywords = {X-ray crystallography}, issn = {0141-8130}, doi = {https://doi.org/10.1016/j.ijbiomac.2018.06.003}, url = {https://www.sciencedirect.com/science/article/pii/S0141813018322384}, author = {Kumar, Krishan and Correia, M{\'a}rcia and Pires, Virg{\'\i}nia R. and Dhillon, Arun and Sharma, Kedar and Rajulapati, Vikky and Fontes, Carlos M. G. A. and Carvalho, Ana Lu{\'\i}sa and Goyal, Arun} } @inbook {10.1039/9781788010641-00159, title = {Targeting protein-carbohydrate interactions in plant cell-wall biodegradation: the power of carbohydrate microarrays}, booktitle = {Carbohydrate Chemistry: Chemical and Biological Approaches Volume 43}, volume = {43}, year = {2018}, note = {n/a}, pages = {159-176}, publisher = {The Royal Society of Chemistry}, organization = {The Royal Society of Chemistry}, abstract = {

The plant cell-wall is constituted by structurally diverse polysaccharides. The biodegradation of these is a crucial process for life sustainability. Cellulolytic microorganisms are highly efficient in this process by assembling modular architectures of carbohydrate-active enzymes with appended non-catalytic carbohydrate-binding modules (CBMs). Carbohydrate microarrays offer high-throughput and sensitive tools for uncovering carbohydrate-binding specificities of CBMs{,} which is pivotal to understand the function of these modules in polysaccharide biodegradation mechanisms. Features of this technology will be here briefly reviewed with highlights of microarray approaches to study plant-carbohydrates and CBM-carbohydrate interactions{,} along with an overview of plant polysaccharides and microorganisms strategies for their recognition.

}, isbn = {978-1-78801-003-0}, doi = {10.1039/9781788010641-00159}, url = {http://dx.doi.org/10.1039/9781788010641-00159}, author = {Ribeiro, Diana O. and Pinheiro, Benedita A. and Carvalho, Ana Luisa and Palma, Angelina S.} } @article {Kryshtafovych2017, title = {Target highlights from the first post-PSI CASP experiment (CASP12, May-August 2016)}, journal = {Proteins: Structure, Function, and Bioinformatics}, year = {2017}, note = {

n/a

}, month = {oct}, abstract = {

The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment. This article is protected by copyright. All rights reserved.

}, keywords = {CASP, NMR, protein structure prediction, X-ray crystallography}, issn = {08873585}, doi = {10.1002/prot.25392}, url = {http://www.ncbi.nlm.nih.gov/pubmed/28960539 http://doi.wiley.com/10.1002/prot.25392}, author = {Kryshtafovych, Andriy and Albrecht, Reinhard and Basl{\'e}, Arnaud and Bule, Pedro and Caputo, Alessandro T. and Carvalho, Ana Luisa and Chao, Kinlin L. and Diskin, Ron and Fidelis, Krzysztof and Fontes, Carlos M. G. A. and Fredslund, Folmer and Gilbert, Harry J. and Goulding, Celia W. and Hartmann, Marcus D. and Hayes, Christopher S. and Herzberg, Osnat and Hill, Johan C. and Joachimiak, Andrzej and Kohring, Gert-Wieland and Koning, Roman I. and {Lo Leggio}, Leila and Mangiagalli, Marco and Michalska, Karolina and Moult, John and Najmudin, Shabir and Nardini, Marco and Nardone, Valentina and Ndeh, Didier and Nguyen, Thanh H. and Pintacuda, Guido and Postel, Sandra and van Raaij, Mark J. and Roversi, Pietro and Shimon, Amir and Singh, Abhimanyu K. and Sundberg, Eric J. and Tars, Kaspars and Zitzmann, Nicole and Schwede, Torsten} } @article {Hussain2017, title = {Tunable gas sensing gels by cooperative assembly}, journal = {Advanced Functional Materials}, volume = {just accep}, number = {xx}, year = {2017}, note = {

n/a

}, month = {may}, pages = {xx}, abstract = {

n/a

}, keywords = {gas sensing, gelatin, ionic liquids, liquid crystals, self-assembly}, issn = {1616301X}, doi = {10.1002/adfm.201700803}, url = {http://doi.wiley.com/10.1002/adfm.201700803}, author = {Hussain, Abid and Semeano, Ana T. S. and Palma, Susana I.C.J. and Pina, Ana S. and Almeida, Jos{\'e} and Medrado, B{\'a}rbara F. and P{\'a}dua, Ana C. C. S. and Carvalho, Ana L. and Dion{\'\i}sio, Madalena and Li, Rosamaria W. C. and Gamboa, Hugo and Ulijn, Rein V. and Gruber, Jonas and Roque, Ana C. A.} } @article {Pires2017a, title = {Stability and ligand promiscuity of type A carbohydrate-binding modules are illustrated by the structure of Spirochaeta thermophila StCBM64C}, journal = {Journal of Biological Chemistry}, volume = {292}, number = {12}, year = {2017}, note = {

n/a

}, month = {mar}, pages = {4847{\textendash}4860}, abstract = {

Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A Carbohydrate-Binding Modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal green fluorescence protein (GFP) domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pHs and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a coplanar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrates how type A CBMs target their appended plant cell wall degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.

}, issn = {1083351X}, doi = {10.1074/jbc.M116.767541}, url = {http://europepmc.org/abstract/med/28179427}, author = {Pires, Virg{\'\i}nia M R and Pereira, Pedro M M and Br{\'a}s, Joana L. A. and Correia, M{\'a}rcia and Cardoso, V{\^a}nia and Bule, Pedro and Alves, Victor D. and Najmudin, Shabir and Venditto, Immacolata and Ferreira, Lu{\'\i}s M A and Rom{\~a}o, Maria Jo{\~a}o and Carvalho, Ana Lu{\'\i}sa and Fontes, Carlos M. G. A. and Prazeres, Duarte Miguel} } @article {Kowacz2017, title = {Infrared light-induced protein crystallization. Structuring of protein interfacial water and periodic self-assembly}, journal = {Journal of Crystal Growth}, volume = {457}, year = {2017}, note = {

n/a

}, month = {jan}, pages = {362{\textendash}368}, abstract = {

We show that a physical trigger, a non-ionizing infrared (IR) radiation at wavelengths strongly absorbed by liquid water, can be used to induce and kinetically control protein (periodic) self-assembly in solution. This phenomenon is explained by considering the effect of IR light on the structuring of protein interfacial water. Our results indicate that the IR radiation can promote enhanced mutual correlations of water molecules in the protein hydration shell. We report on the radiation-induced increase in both the strength and cooperativeness of H-bonds. The presence of a structured dipolar hydration layer can lead to attractive interactions between like-charged biomacromolecules in solution (and crystal nucleation events). Furthermore, our study suggests that enveloping the protein within a layer of structured solvent (an effect enhanced by IR light) can prevent the protein non-specific aggregation favoring periodic self-assembly. Recognizing the ability to affect protein-water interactions by means of IR radiation may have important implications for biological and bio-inspired systems.

}, keywords = {A1. Protein crystallization, A2. Protein hydration, A3. Colloidal self-assembly, A4. Crystal growth from solution}, issn = {00220248}, doi = {10.1016/j.jcrysgro.2016.01.003}, url = {http://linkinghub.elsevier.com/retrieve/pii/S0022024816000178}, author = {Kowacz, Magdalena and Marchel, Mateusz and Juknait{\'e}, Lina and Esperan{\c c}a, Jos{\'e} M S S and Rom{\~a}o, Maria Jo{\~a}o and Carvalho, Ana Lu{\'\i}sa and Rebelo, Lu{\'\i}s Paulo N.} } @article {11024, title = {Assembly of Ruminococcus flavefaciens cellulosome revealed by structures of two cohesin-dockerin complexes}, journal = {Scientific Reports}, volume = {7}, year = {2017}, note = {

n/a

}, month = {2017}, pages = {759}, abstract = {

Cellulosomes are sophisticated multi-enzymatic nanomachines produced by anaerobes to effectively deconstruct plant structural carbohydrates. Cellulosome assembly involves the binding of enzyme-borne dockerins (Doc) to repeated cohesin (Coh) modules located in a non-catalytic scaffoldin. Docs appended to cellulosomal enzymes generally present two similar Coh-binding interfaces supporting a dual-binding mode, which may confer increased positional adjustment of the different complex components. Ruminococcus flavefaciens{\textquoteright} cellulosome is assembled from a repertoire of 223 Doc-containing proteins classified into 6 groups. Recent studies revealed that Docs of groups 3 and 6 are recruited to the cellulosome via a single-binding mode mechanism with an adaptor scaffoldin. To investigate the extent to which the single-binding mode contributes to the assembly of R. flavefaciens cellulosome, the structures of two group 1 Docs bound to Cohs of primary (ScaA) and adaptor (ScaB) scaffoldins were solved. The data revealed that group 1 Docs display a conserved mechanism of Coh recognition involving a single-binding mode. Therefore, in contrast to all cellulosomes described to date, the assembly of R. flavefaciens cellulosome involves single but not dual-binding mode Docs. Thus, this work reveals a novel mechanism of cellulosome assembly and challenges the ubiquitous implication of the dual-binding mode in the acquisition of cellulosome flexibility.

}, isbn = {2045-2322}, url = {http://dx.doi.org/10.1038/s41598-017-00919-w}, author = {Bule, Pedro and Alves, Victor D. and Israeli-Ruimy, Vered and Carvalho, Ana L. and Ferreira, Lu{\'\i}s M A and Smith, Steven P. and Gilbert, Harry J. and Najmudin, Shabir and Bayer, Edward A. and Fontes, Carlos M. G. A.} } @article {11331, title = {Ion-Exchange Membranes for Stable Derivatization of Protein Crystals}, journal = {Crystal Growth \& DesignCrystal Growth \& Design}, year = {2017}, note = {

doi: 10.1021/acs.cgd.7b00315

}, month = {2017}, publisher = {American Chemical Society}, abstract = {n/a}, isbn = {1528-7483}, url = {http://dx.doi.org/10.1021/acs.cgd.7b00315}, author = {Polino, Mariella and Carvalho, Ana Lu{\i}́sa and Juknaite, Lina and Portugal, Carla A. M. and Coelhoso, Isabel M. and Rom{\~a}o, Maria Jo{\~a}o and Crespo, Jo{\~a}o G} } @article {dosSantos2016, title = {Renaissance of protein crystallization and precipitation in biopharmaceuticals purification}, journal = {Biotechnology Advances}, year = {2017}, note = {

n/a

}, pages = {-}, abstract = {

Abstract The current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups.

}, keywords = {precipitation}, issn = {0734-9750}, doi = {http://dx.doi.org/10.1016/j.biotechadv.2016.11.005}, url = {http://www.sciencedirect.com/science/article/pii/S0734975016301513}, author = {Raquel dos Santos and Carvalho, Ana Lu{\'\i}sa and A. Cec{\'\i}lia A. Roque} } @article {Bras2016, title = {Diverse specificity of cellulosome attachment to the bacterial cell surface}, journal = {Scientific Reports}, volume = {6}, year = {2016}, note = {

n/a

}, month = {dec}, pages = {38292}, publisher = {The Author(s)}, abstract = {

During the course of evolution, the cellulosome, one of Nature{\textquoteright}s most intricate multi-enzyme complexes, has been continuously fine-tuned to efficiently deconstruct recalcitrant carbohydrates. To facilitate the uptake of released sugars, anaerobic bacteria use highly ordered protein-protein interactions to recruit these nanomachines to the cell surface. Dockerin modules located within a non-catalytic macromolecular scaffold, whose primary role is to assemble cellulosomal enzymatic subunits, bind cohesin modules of cell envelope proteins, thereby anchoring the cellulosome onto the bacterial cell. Here we have elucidated the unique molecular mechanisms used by anaerobic bacteria for cellulosome cellular attachment. The structure and biochemical analysis of five cohesin-dockerin complexes revealed that cell surface dockerins contain two cohesin-binding interfaces, which can present different or identical specificities. In contrast to the current static model, we propose that dockerins utilize multivalent modes of cohesin recognition to recruit cellulosomes to the cell surface, a mechanism that maximises substrate access while facilitating complex assembly.

}, url = {https://www.nature.com/articles/srep38292}, author = {Br{\'a}s, Joana L. A. and Pinheiro, Benedita A. and Cameron, Kate and Cuskin, Fiona and Viegas, Aldino and Najmudin, Shabir and Bule, Pedro and Pires, Virginia M. R. and Rom{\~a}o, Maria Jo{\~a}o and Bayer, Edward A. and Spencer, Holly L. and Smith, Steven and Gilbert, Harry J. and Alves, Victor D. and Carvalho, Ana Lu{\'\i}sa and Fontes, Carlos M. G. A.} } @article {9898, title = {Ionic-Liquid-Functionalized Mineral Particles for Protein Crystallization}, journal = {Crystal Growth \& Design}, volume = {15}, number = {6}, year = {2015}, note = {

ISI Document Delivery No.: CK0JB Times Cited: 0 Cited Reference Count: 42 Kowacz, M. Marchel, M. Juknaite, L. Esperanca, J. M. S. S. Romao, M. J. Carvalho, A. L. Rebelo, L. P. N. Esperanca, Jose/B-5116-2008; Esperanca, Jose/0000-0001-9615-8678; Marchel, Mateusz/0000-0002-2274-1139; Juknaite, Lina/0000-0002-5739-7788 Fundacao para a Ciencia e a Tecnologia (FCT), Portugal [PTDC/BBB-BEP/3058/2012, PEst-OE/EQB/LA0004/2013, UID/Multi/04378/2013, PEst-C/EQB/LA0006/2013, RECI/BBB-BEP/0124/2012, SFRH/BPD/63554/2009]; Investigator FCT Program; COMPETE Program The authors thank Dr. Teresa Santos-Silva and Ph.D. student Hugo Correia for kindly providing the crystallization conditions for bovine blood hemoglobin and pancreatic trypsin. The authors acknowledge financial support from Fundacao para a Ciencia e a Tecnologia (FCT), Portugal, through R\&D Projects PTDC/BBB-BEP/3058/2012, PEst-OE/EQB/LA0004/2013 (to ITQB), UID/Multi/04378/2013, PEst-C/EQB/LA0006/2013 (to Associate Lab UCIBIO-REQUIMTE), and RECI/BBB-BEP/0124/2012 and also through a postdoctoral grant (SFRH/BPD/63554/2009) and a contract under the Investigator FCT 2012 Program and the COMPETE Program. The authors also thank the ESRF (Grenoble, France) and the Diamond Light Source (Didcot, U.K.) for access to data collection facilities. 9 Amer chemical soc Washington 1528-7505

}, pages = {2994-3003}, type = {Journal Article}, abstract = {

Nucleation is a critical step determining the outcome of the entire crystallization process. Finding an effective nucleant for protein crystallization is of utmost importance for structural biology. The latter relies on good-quality crystals to solve the three-dimensional structures of macromolecules. In this study we show that crystalline barium sulfate (BaSO4) with an etched and/or ionic liquid (IL)-functionalized surface (1) can induce protein nucleation at concentrations well below the concentration needed to promote crystal growth under control conditions, (2) can shorten the nucleation time, (3) can increase the growth rate, and finally (4) may help to improve the protein crystal morphology. These effects were shown for lysozyme, RNase A, trypsin, proteinase K, myoglobin, and hemoglobin. Therefore, the use of BaSO4 particles enables us to reduce the amount of protein in crystallization trials and increases the chance of obtaining protein crystals of the desired quality. In the context of the underlying mechanism, it is shown that the protein-solid contact formation is governed by the interaction of the polar compartments of the biomacromolecule with the support. The tendency of a protein to concentrate near the solid surface is enhanced by both the hydrophobicity of the protein and that of the surface (tuned by the functionalizing IL). These mechanisms of interaction of biomacromolecules with inorganic hydrophilic solids correspond to the principles of amphiphilic IL-mineral interactions.

}, keywords = {heterogeneous nucleation crystal nucleation adsorption stability surfaces water lysozyme fluorescence polystyrene suspensions Chemistry Crystallography Materials Science}, issn = {1528-7483}, doi = {10.1021/acs.cgd.5b00403}, url = {://WOS:000355890400056}, author = {Kowacz, M. and Marchel, M. and Juknaite, L. and Esperanca, Jmss and Romao, MJ and Carvalho, AL and Rebelo, L. P. N.} } @article {8484, title = {Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry}, journal = {Mol Cell Proteomics}, year = {2015}, note = {

n/a

}, type = {Journal Article}, abstract = {

Glucans are polymers of D-glucose with differing linkages in linear or branched sequences. They are constituents of microbial and plant cell-walls and involved in important bio-recognition processes including immunomodulation, anti-cancer activities, pathogen virulence and plant cell-wall biodegradation. Translational possibilities for these activities in medicine and biotechnology are considerable. High-throughput micro-methods are needed to screen proteins for recognition of specific glucan sequences as a lead to structure-function studies and their exploitation. We describe construction of a glucome microarray, the first sequence-defined glycome-scale microarray, using a designer approach from targeted ligand-bearing glucans in conjunction with a novel high-sensitivity mass spectrometric sequencing method, as a screening tool to assign glucan recognition motifs. The glucome microarray comprises 153 oligosaccharide probes with high purity, representing major sequences in glucans. The negative-ion electrospray tandem mass spectrometry with collision-induced dissociation was used for complete linkage analysis of gluco-oligosaccharides in linear homo and hetero and branched sequences. The system is validated using antibodies and carbohydrate-binding modules known to target α- or β-glucans in different biological contexts, extending knowledge on their specificities, and applied to reveal new information on glucan recognition by two signalling molecules of the immune system against pathogens: Dectin-1 and DC-SIGN. The sequencing of the glucan oligosaccharides by the MS method and their interrogation on the microarrays provides detailed information on linkage, sequence and chain length requirements of glucan-recognizing proteins, and are a sensitive means of revealing unsuspected sequences in the polysaccharides.

}, issn = {1535-9484}, doi = {10.1074/mcp.M115.048272}, url = {http://www.ncbi.nlm.nih.gov/pubmed/25670804}, author = {Palma, A. S. and Liu, Y. and Zhang, H. and Zhang, Y and McCleary, B. V. and Yu, G. and Huang, Q. and Guidolin, L. S. and Ciocchini, A. E. and Torosantucci, A. and Wang, D. and Carvalho, AL and Fontes, C. M. and Mulloy, B. and Childs, R. A. and Feizi, T. and Chai, W.} } @article {6012, title = {Use of Gold Nanoparticles as Additives in Protein Crystallization}, journal = {Crystal Growth \& Design}, volume = {14}, number = {1}, year = {2014}, note = {

Times Cited: 1 Carvalho, Ana Luisa/G-5638-2011; Romao, Maria/A-4115-2013; Pereira, Eulalia/C-6282-2013 Carvalho, Ana Luisa/0000-0002-3824-0240; Romao, Maria/0000-0002-3004-0543; Pereira, Eulalia/0000-0003-2086-5696 1

}, pages = {222-227}, type = {Journal Article}, abstract = {n/a}, issn = {1528-7483; 1528-7505}, doi = {10.1021/cg4014398}, url = {://WOS:000329337000029}, author = {Ribeiro, Diana and Kulakova, Alina and Quaresma, Pedro and Pereira, Eulalia and Bonifacio, Cecilia and Romao, Maria Joao and Franco, Ricardo and Carvalho, Ana Luisa} } @article {4840, title = {Solution structure, dynamics and binding studies of a family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11)}, journal = {The Biochemical journal}, volume = {451}, number = {2}, year = {2013}, note = {

23356867

}, pages = {289-300}, type = {Journal Article}, issn = {1470-8728}, doi = {10.1042/bj20120627}, url = {://MEDLINE:23356867}, author = {Viegas, Aldino and Sardinha, Joao and Freire, Filipe and Duarte, Daniel F and Carvalho, Ana L. and Fontes, Carlos M. G. A. and Romao, Maria J. and Macedo, Anjos L and Cabrita, Eurico J.} } @inbook {4836, title = {Escherichia coli expression, purification, crystallization, and structure determination of bacterial cohesin-dockerin complexes}, booktitle = {Methods in enzymology}, volume = {510}, year = {2012}, note = {

22608738

}, pages = {395-415}, type = {Journal Article}, abstract = {

Cellulosomes are highly efficient nanomachines that play a fundamental role during the anaerobic deconstruction of complex plant cell wall carbohydrates. The assembly of these complex nanomachines results from the very tight binding of repetitive cohesin modules, located in a noncatalytic molecular scaffold, and dockerin domains located at the C-terminus of the enzyme components of the cellulosome. The number of enzymes found in a cellulosome varies but may reach more than 100 catalytic subunits if cellulosomes are further organized in polycellulosomes, through a second type of cohesin-dockerin interaction. Structural studies have revealed how the cohesin-dockerin interaction mediates cellulosome assembly and cell-surface attachment, while retaining the flexibility required to potentiate catalytic synergy within the complex. Methods that might be applied for the production, purification, and structure determination of cohesin-dockerin complexes are described here. Copyright 2012 Elsevier Inc. All rights reserved.

}, issn = {1557-7988}, url = {://MEDLINE:22608738}, author = {Bras, Joana L. A. and Carvalho, Ana Luisa and Viegas, Aldino and Najmudin, Shabir and Alves, Victor D. and Prates, Jose A. M. and Ferreira, Luis M. A. and Romao, Maria J. and Gilbert, Harry J. and Fontes, Carlos M. G. A.} } @article {4839, title = {Flexibility and specificity of the cohesin-dockerin interaction: implications for cellulosome assembly and functionality}, journal = {Biocatalysis and Biotransformation}, volume = {30}, number = {3}, year = {2012}, note = {

ISI Document Delivery No.: 951WJ Times Cited: 0 Cited Reference Count: 45 Pinheiro, Benedita Andrade Armada Bras, Joana Luis Najmudin, Shabir Carvalho, Ana Luisa Ferreira, Luis M. A. Prates, Jose A. M. Godinho Andrade Fontes, Carlos Mendes INFORMA HEALTHCARE LONDON

}, pages = {309-315}, type = {Journal Article}, abstract = {

Cellulosomes are highly elaborate multi-enzyme complexes of Carbohydrate Active enZYmes (CAZYmes) secreted by cellulolytic microorganisms, which very effectively degrade the most abundant polymers on Earth, cellulose and hemicelluloses. Cellulosome assembly requires that a non-catalytic dockerin module found in cellulosomal enzymes binds to one of the various cohesin domains located in a large molecular scaffold called Scaffoldin. A diversity of cohesin -dockerin binding specificities have been described, the combination of which may result in complex plant cell wall degrading systems, maximising the synergy between enzymes in order to improve catalytic efficiency. Structural studies have allowed the spatial flexibility inherent to the cellulosomal system to be determined. Recent progress achieved from the study of the fundamental cohesin and dockerin units involved in cellulosome assembly will be reviewed.

}, issn = {1024-2422}, doi = {10.3109/10242422.2012.681854}, url = {://WOS:000304750500005}, author = {Pinheiro, B. A. and Bras, J. L. A. and Najmudin, S. and Carvalho, AL and Ferreira, LMA and Prates, JAM and Fontes, CMGA} } @article {4837, title = {Hofmeister effects of ionic liquids in protein crystallization: Direct and water-mediated interactions}, journal = {Crystengcomm}, volume = {14}, number = {15}, year = {2012}, note = {

Times Cited: 0

}, pages = {4912-4921}, type = {Journal Article}, abstract = {n/a}, issn = {1466-8033}, doi = {10.1039/c2ce25129a}, url = {://WOS:000305999500006}, author = {Kowacz, Magdalena and Mukhopadhyay, Abhik and Carvalho, Ana Luisa and Esperanca, Jose M. S. S. and Romao, Maria J. and Luis Paulo N. Rebelo} } @article {4835, title = {Novel Clostridium thermocellum Type I Cohesin-Dockerin Complexes Reveal a Single Binding Mode}, journal = {The Journal of biological chemistry}, volume = {287}, number = {53}, year = {2012}, note = {

23118225

}, pages = {44394-405}, type = {Journal Article}, issn = {1083-351X}, doi = {10.1074/jbc.M112.407700}, url = {://MEDLINE:23118225}, author = {Bras, Joana L. A. and Alves, Victor D. and Carvalho, Ana Luisa and Najmudin, Shabir and Prates, Jose A. M. and Ferreira, Luis M. A. and Bolam, David N. and Romao, Maria Joao and Gilbert, Harry J. and Fontes, Carlos M. G. A.} } @article {1808, title = {Molecular Architecture and Structural Transitions of a Clostridium thermocellum Mini-Cellulosome}, journal = {Journal of Molecular Biology}, volume = {407}, number = {4}, year = {2011}, note = {n/a}, pages = {571-580}, abstract = {n/a}, author = {Garcia-Alvarez, Begona and Melero, Roberto and Dias, Fernando M. V. and Prates, Jose A. M. and Fontes, Carlos M. G. A. and Smith, Steven P. and Romao, Maria Joao and Carvalho, Ana Luisa and Llorca, Oscar} } @article {1797, title = {Structural insights into a unique cellulase fold and mechanism of cellulose hydrolysis (vol 108, pg 5237, 2011)}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {108}, number = {20}, year = {2011}, note = {

Times Cited: 0

}, pages = {8525-8525}, abstract = {n/a}, author = {Bras, Joana L. A. and Cartmell, Alan and Carvalho, Ana Lusia M. and Verze, Genny and Bayer, Edward A. and Vazana, Yael and Correia, Marcia A. S. and Prates, Jose A. M. and Ratnaparkhe, Supriya and Boraston, Alisdair B. and Romao, Maria J. and Fontes, Carlos M. G. A. and Gilbert, Harry J.} } @inbook {Carvalho2010, title = {X-Ray Crystallography in Drug Discovery}, booktitle = {Methods in molecular biology (Clifton, N.J.)}, volume = {572}, year = {2010}, note = {

n/a

}, pages = {31{\textendash}56}, abstract = {

Macromolecular X-ray crystallography is an important and powerful technique in drug discovery, used by pharmaceutical companies in the discovery process of new medicines. The detailed analysis of crystal structures of protein-ligand complexes allows the study of the specific interactions of a particular drug with its protein target at the atomic level. It is used to design and improve drugs. The starting point of these studies is the preparation of suitable crystals of complexes with potential ligands, which can be achieved by using different strategies described in this chapter. In addition, an introduction to X-ray crystallography is given, highlighting the fundamental steps necessary to determine the three-dimensional structure of protein-ligand complexes, as well as some of the tools and criteria to validate crystal structures available in databases.

}, issn = {1940-6029}, doi = {10.1007/978-1-60761-244-5_3}, url = {http://www.scopus.com/inward/record.url?eid=2-s2.0-79952112755{\&}partnerID=MN8TOARS http://www.ncbi.nlm.nih.gov/pubmed/20694684{\%}0Ahttp://link.springer.com/10.1007/978-1-60761-244-5{\_}3}, author = {Carvalho, Ana Lu{\'\i}sa and Trinc{\~a}o, Jos{\'e} and Rom{\~a}o, Maria Jo{\~a}o} } @article {1809, title = {Liquid crystalline cellulose derivative elastomer films under uniaxial strain}, journal = {Cellulose}, volume = {16}, number = {2}, year = {2009}, note = {n/a}, pages = {199-205}, abstract = {n/a}, author = {Godinho, M. H. and Filip, D. and Costa, I. and Carvalho, A. -L. and Figueirinhas, J. L. and Terentjev, E. M.} } @article {1807, title = {Preliminary structural characterization of human SOUL, a haem-binding protein}, journal = {Acta Crystallographica Section F-Structural Biology and Crystallization Communications}, volume = {65}, year = {2009}, note = {n/a}, pages = {723-726}, abstract = {n/a}, author = {Freire, Filipe and Romao, Maria Joao and Macedo, Anjos L and Aveiro, Susana S. and Goodfellow, Brian J. and Carvalho, Ana Luisa} } @article {1815, title = {Real-Time Monitoring of Molecular Dynamics of Ethylene Glycol Dimethacrylate Glass Former}, journal = {Journal of Physical Chemistry B}, volume = {113}, number = {43}, year = {2009}, note = {n/a}, pages = {14209-14217}, abstract = {n/a}, author = {Viciosa, M. T. and N. T. Correia and Salmeron Sanchez, M. and Carvalho, AL and Romao, MJ and Gomez Ribelles, J. L. and Dionisio, M.} } @article {1816, title = {Molecular determinants of ligand specificity in family 11 carbohydrate binding modules - an NMR, X-ray crystallography and computational chemistry approach}, journal = {Febs Journal}, volume = {275}, number = {10}, year = {2008}, note = {n/a}, pages = {2524-2535}, abstract = {n/a}, author = {Viegas, Aldino and Bras, Natercia F. and Cerqueira, Nuno M. F. S. A. and Fernandes, Pedro Alexandrino and Prates, Jose A. M. and Fontes, Carlos M. G. A. and Bruix, Marta and Romao, Maria Joao and Carvalho, Ana Luisa and Ramos, Maria Joao and Macedo, Anjos L and Cabrita, Eurico J.} } @article {1804, title = {Evidence for a dual binding mode of dockerin modules to cohesins}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {104}, number = {9}, year = {2007}, note = {n/a}, pages = {3089-3094}, abstract = {n/a}, author = {Carvalho, Ana Luisa and Dias, Fernando M. V. and Nagy, Tibor and Prates, Jose A. M. and Proctor, Mark R. and Smith, Nicola and Bayer, Edward A. and Davies, Gideon J. and Ferreira, Luis M. A. and Romao, Maria J. and Fontes, Carlos M. G. A. and Gilbert, Harry J.} } @article {1805, title = {Heterodimeric nitrate reductase (NapAB) from Cupriavidus necator H16: purification, crystallization and preliminary X-ray analysis}, journal = {Acta Crystallographica Section F-Structural Biology and Crystallization Communications}, volume = {63}, year = {2007}, note = {n/a}, pages = {516-519}, abstract = {n/a}, author = {Coelho, Catarina and Gonzalez, Pablo J. and Trincao, Jose and Carvalho, Ana L. and Najmudin, Shabir and Hettman, Thomas and Dieckman, Stephan and Moura, Jose J. G. and Moura, Isabel and Romao, Maria J.} } @article {1814, title = {The first crystal structure of class III superoxide reductase from Treponema pallidum}, journal = {Journal of Biological Inorganic Chemistry}, volume = {11}, number = {5}, year = {2006}, note = {n/a}, pages = {548-558}, abstract = {n/a}, author = {Santos-Silva, T and Trincao, J and Carvalho, AL and Bonifacio, C. and Auchere, F and Raleiras, P and Moura, I and Moura, JJG and Romao, MJ} } @article {1810, title = {Xyloglucan is recognized by carbohydrate-binding modules that interact with beta-glucan chains}, journal = {Journal of Biological Chemistry}, volume = {281}, number = {13}, year = {2006}, note = {n/a}, pages = {8815-8828}, abstract = {n/a}, author = {Najmudin, S. and Guerreiro, CIPD and Carvalho, AL and Prates, JAM and Correia, MAS and Alves, V. D. and Ferreira, LMA and Romao, MJ and Gilbert, HJ and Bolam, DN and Fontes, CMGA} } @article {1802, title = {Insights into the structural determinants of cohesin dockerin specificity revealed by the crystal structure of the type II cohesin from Clostridium thermocellum SdbA}, journal = {Journal of Molecular Biology}, volume = {349}, number = {5}, year = {2005}, note = {n/a}, pages = {909-915}, abstract = {n/a}, author = {Carvalho, AL and Pires, VMR and Gloster, TM and Turkenburg, JP and Prates, JAM and Ferreira, LMA and Romao, MJ and Davies, GJ and Fontes, CMGA and Gilbert, HJ} } @article {1813, title = {Superoxide reductase from the syphilis spirochete Treponema pallidum: crystallization and structure determination using soft X-rays}, journal = {Acta Crystallographica Section F-Structural Biology and Crystallization Communications}, volume = {61}, year = {2005}, note = {n/a}, pages = {967-970}, abstract = {n/a}, author = {Santos-Silva, T and Trincao, J and Carvalho, AL and Bonifacio, C. and Auchere, F and Moura, I and Moura, JJG and Romao, MJ} } @article {1801, title = {The family 11 carbohydrate-binding module of Clostridium thermocellum Lic26A-Cel5E accommodates beta-1,4- and beta-1,3-1,4-mixed linked glucans at a single binding site}, journal = {Journal of Biological Chemistry}, volume = {279}, number = {33}, year = {2004}, note = {n/a}, pages = {34785-34793}, abstract = {n/a}, author = {Carvalho, AL and Goyal, A and Prates, JAM and Bolam, DN and Gilbert, HJ and Pires, VMR and Ferreira, LMA and Planas, A and Romao, MJ and Fontes, CMGA} } @article {1799, title = {Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {100}, number = {24}, year = {2003}, note = {n/a}, pages = {13809-13814}, abstract = {n/a}, author = {Carvalho, AL and Dias, FMV and Prates, JAM and Nagy, T and Gilbert, HJ and Davies, GJ and Ferreira, LMA and Romao, MJ and Fontes, CMGA} } @article {1803, title = {Crystal structure of a prostate kallikrein isolated from stallion seminal plasma: A homologue of human PSA}, journal = {Journal of Molecular Biology}, volume = {322}, number = {2}, year = {2002}, note = {n/a}, pages = {325-337}, abstract = {n/a}, author = {Carvalho, AL and Sanz, L. and Barettino, D and Romero, A. and Calvete, JJ and Romao, MJ} } @article {1798, title = {Purification, crystallization and identification by X-ray analysis of a prostate kallikrein from horse seminal plasma}, journal = {Acta Crystallographica Section D-Biological Crystallography}, volume = {57}, year = {2001}, note = {

ISI Document Delivery No.: 455RV Times Cited: 2 Cited Reference Count: 31 Carvalho, AL Dias, JM Sanz, L Romero, A Calvete, JJ Romao, MJ MUNKSGAARD INT PUBL LTD COPENHAGEN Part 8

}, pages = {1180-1183}, abstract = {

The purification, crystallization and identification by X-ray diffraction analysis of a horse kallikrein is reported. The protein was purired from horse seminal plasma. Crystals belong to space group C2 and the structure was solved by the MIRAS method, with two heavy-atom derivatives of mercury and platinum. X-ray diffraction data to 1.42 Angstrom resolution were collected at the ESRF synchrotron-radiation source.

}, keywords = {CRYSTAL-STRUCTURE SERINE PROTEINASE BOVINE TRYPSIN SEMENOGELIN-I HUMAN-SEMEN ANTIGEN EXPRESSION FAMILY GENE REFINEMENT}, author = {Carvalho, AL and Dias, JM and Sanz, L. and Romero, A. and Calvete, JJ and Romao, MJ} } @article {1796, title = {Structural studies by X-ray diffraction on metal substituted desulforedoxin, a rubredoxin-type protein}, journal = {Protein Science}, volume = {8}, number = {7}, year = {1999}, note = {n/a}, pages = {1536-1545}, abstract = {n/a}, author = {Archer, M and Carvalho, AL and Teixeira, S and Moura, I and Moura, JJG and Rusnak, F and Romao, MJ} } @article {1811, title = {Crystal structure of acidic seminal fluid protein (aSFP) at 1.9 angstrom resolution: a bovine polypeptide of the spermadhesin family}, journal = {Journal of Molecular Biology}, volume = {274}, number = {4}, year = {1997}, note = {

ISI Document Delivery No.: YP198 Times Cited: 33 Cited Reference Count: 32 Romao, MJ Kolln, I Dias, JM Carvalho, AL Romero, A Varela, PF Sanz, L Topfer-Petersen, E Calvete, JJ ACADEMIC PRESS LTD LONDON

}, pages = {650-660}, abstract = {

We report the three-dimensional crystal structure of acidic seminal fluid protein (aSFP), a 12.9 kDa poly-peptide of the spermadhesin family isolated from bovine seminal plasma, solved by the multiple isomorphous replacement method and refined with data to 1.9 Angstrom resolution with a final R-factor of 17.3\%. aSFP is built by a single CUB domain architecture, a 100 to 110 amino-acid-residue extracellular module found in 16 functionally diverse proteins. The structure of aSFP reveals that the CUB domain displays a beta-sandwich topology organised into two 5-stranded beta-sheets, each of which contain two parallel and four antiparallel strands. The structure of aSFP is almost identical to that of porcine spermadhesins PSP-I and PSP-II, which in turn show limited structural similarity with jellyroll topologies of certain virus capsid proteins. Essentially, topologically conserved residues in these proteins are those internal amino acids forming the hydrophobic core of the CUB and the jellyroll domains, suggesting their importance in maintaining the integrity of these protein folds, On the other hand, the structure of aSFP shows structural features that are unique to this protein and which may provide a structural ground for understanding the distinct biological properties of different members of the spermadhesin protein family. (C) 1997 Academic Press Limited.

}, keywords = {bovine seminal plasma protein acidic seminal fluid protein aSFP spermadhesins CUB domain PELLUCIDA BINDING CHARACTERISTICS BOAR SPERMADHESINS PLASMA OLIGOSACCHARIDE LOCALIZATION LOCATION}, author = {Romao, MJ and Kolln, I. and Dias, JM and Carvalho, AL and Romero, A. and Varela, P. F. and Sanz, L. and Topfer-Petersen, E. and Calvete, JJ} } @article {1812, title = {The crystal structures of two spermadhesins reveal the CUB domain fold}, journal = {Nature Structural Biology}, volume = {4}, number = {10}, year = {1997}, note = {

ISI Document Delivery No.: YA203 Times Cited: 108 Cited Reference Count: 32 Romero, A Romao, MJ Varela, PF Kolln, I Dias, JM Carvalho, AL Sanz, L TopferPetersen, E Calvete, JJ NATURE PUBLISHING CO NEW YORK

}, pages = {783-788}, abstract = {

Spermadhesins, 12,000-14,000 M-r mammalian proteins, include lectins involved in sperm-egg binding and display a single CUB domain architecture. We report the crystal structures of porcine seminal plasma PSP-I/PSP-II, a heterodimer of two glycosylated spermadhesins. and bovine aSFP at 2.4 Angstrom and 1.9 Angstrom resolution respectively.

}, keywords = {SEMINAL FLUID PROTEIN BOAR SPERMADHESINS PSP-II PLASMA HETERODIMER LOCATION CLONING RNA}, author = {Romero, A. and Romao, MJ and Varela, P. F. and Kolln, I. and Dias, JM and Carvalho, AL and Sanz, L. and TopferPetersen, E. and Calvete, JJ} } @article {1806, title = {Crystallization and preliminary x-ray diffraction studies of aSFP, a bovine seminal plasma protein with a single CUB domain architecture}, journal = {Protein Science}, volume = {6}, number = {3}, year = {1997}, note = {

ISI Document Delivery No.: WM740 Times Cited: 7 Cited Reference Count: 22 Dias, JM Carvalho, AL Kolln, I Calvete, JJ TopferPetersen, E Varela, PF Romero, A Urbanke, C Romao, MJ CAMBRIDGE UNIV PRESS NEW YORK

}, pages = {725-727}, abstract = {

{Bovine acidic seminal fluid protein (aSFP) is a 12.9 kDa polypeptide of the spermadhesin family built by a single CUB domain architecture. The CUB domain is an extracellular module present in 16 functionally diverse proteins. To determine the three-dimensional structure of aSFP, the protein was crystallized at 21 degrees C by vapor diffusion in hanging drops, using ammonium sulfate, pH 4.7, and polyethyleneglycol 4000 as precipitants, containing 10\% dioxane to avoid the formation of clustered crystals. Elongated prismatic crystals with maximal size of 0.6 x 0.3 x 0.2 mm(3) diffract to beyond 1.9 Angstrom resolution and belong to space group P2(1)2(1)2, with cell parameters a = 52.4 Angstrom

}, keywords = {acidic seminal fluid protein analytical ultracentrifugation aSFP bovine seminal plasma crystallization CUB domain spermadhesin protein family X-ray diffraction analysis FLUID PROTEIN BOAR SPERMADHESINS ZONA-PELLUCIDA LOCALIZATION AWN-1}, author = {Dias, JM and Carvalho, AL and Kolln, I. and Calvete, JJ and TopferPetersen, E. and Varela, P. F. and Romero, A. and Urbanke, C. and Romao, MJ} }