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2020
dos Santos, Raquel, Inês Iria, Ana M. Manuel, Ana P. Leandro, Catarina A. C. Madeira, Joao Goncalves, Ana Luísa Carvalho, and Ana Cecília Roque. "Magnetic Precipitation: A New Platform for Protein Purification." Biotechnology JournalBiotechnology Journal. n/a.n/a (2020): 2000151. AbstractWebsite

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°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

Gomes, Ana Sara, Helena Ramos, Sara Gomes, Joana B. Loureiro, Joana Soares, Valentina Barcherini, Paola Monti, Gilberto Fronza, Carla Oliveira, Lucília Domingues, Margarida Bastos, Daniel F. A. R. Dourado, Ana Luísa Carvalho, Maria João Romão, Benedita Pinheiro, Filipa Marcelo, Alexandra Carvalho, Maria M. M. Santos, and Lucília Saraiva. "SLMP53-1 interacts with wild-type and mutant p53 DNA-binding domain and reactivates multiple hotspot mutations." 1864.1 (2020): 129440. AbstractWebsite

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.

Outis, Mani, Vitor Rosa, César AT Laia, João Carlos Lima, Sónia Barroso, Maria José Calhorda, Ana Luísa Carvalho, and Maria Teresa Aviles Perea. "Synthesis, Crystal Structure and DFT Study of Two New Dinuclear Copper(I) Complexes Bearing Ar-BIAN ligands Funtionalized with NO2 Groups." European Journal of Inorganic ChemistryEuropean Journal of Inorganic Chemistry. n/a.n/a (2020). AbstractWebsite

Two new bis(aryl-imino)-acenaphthene, Ar-BIAN (Ar = 2,4,6-trimethylphenyl = mes) ligands, bearing the NO 2 group in the naphthalene moiety of the iminoacenaphtene at para- (5-NO 2 ) and meta- (4-NO 2 ) position, of formulations 1,2-bis(mes-imino)-5-nitroacenaphthene, 1 , and 1,2-bis(mes-imino)-4-nitroacenaphthene, 2 , were synthesized. Their respective dinuclear iodide bridged copper(I) complexes [Cu 2 (?-I) 2 (mes-BIAN-5-NO 2 ) 2 ], 3 and [Cu 2 (?-I) 2 (mes-BIAN-4-NO 2 ) 2 ] , 4 , were obtained in good yields by treatment with an equimolar amount of CuI. All compounds were characterized by elemental analysis, single crystal X-ray diffraction, 1 H-NMR, 13 C-NMR, FTIR, UV-Vis spectroscopy. DFT calculations helped to understand the different molecular structure observed in the crystals of 3 and 4 and the determining role of packing forces. TDDFT revealed that the absorption bands in the visible were essentially MLCT (Metal to Ligand Charge Transfer), with some n?π* character (intra ligand). The shift to the red compared to the spectrum of the Cu(I) complex analogue without the NO 2 group, [Cu 2 (?-I) 2 ](mes-BIAN) 2 ], 6 , could be explained by the stabilization of the ligand unoccupied π* orbitals in the presence of NO 2 .

2019
Ribeiro, Diana O., Aldino Viegas, Virgínia M. R. Pires, João Medeiros-Silva, Pedro Bule, Wengang Chai, Filipa Marcelo, Carlos M. G. A. Fontes, Eurico J. Cabrita, Angelina S. Palma, and Ana Luísa Carvalho. "Molecular basis for the preferential recognition of β1,3-1,4-glucans by the family 11 carbohydrate-binding module from Clostridium thermocellum." The FEBS JournalThe FEBS Journal. n/a.n/a (2019). AbstractWebsite

Understanding the specific molecular interactions between proteins and ?1,3-1,4-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 ?1,3-1,4-mixed-linked over ?1,4-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 ?1,3-1,4-linked glucose oligosaccharides showed that both the chain length and the position of the ?1,3-linkage are important for recognition, and identified the tetrasaccharide Glc?1,4Glc?1,4Glc?1,3Glc 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 ?1,3-1,4-mixed-linked glucans. This is mediated by a conformation?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 ?1,3-linkage at the reducing end and at specific positions along the ?1,4-linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed-linked ?-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.

Peixoto, Daniela, Gabriela Malta, Hugo Cruz, Sónia Barroso, Ana Luísa Carvalho, Luísa M. Ferreira, and Paula S. Branco. "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." The Journal of Organic ChemistryThe Journal of Organic Chemistry (2019). AbstractWebsite
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Correia, Viviana G., Benedita A. Pinheiro, Ana Luísa Carvalho, and Angelina S. Palma. "Resistance to Aminoglycosides." Antibiotic Drug Resistance. John Wiley & Sons, Ltd, 2019. 1-38. 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.

2018
Carvalho, Ana Luísa, Teresa Santos-Silva, Maria João Romão, J. Eurico, and Filipa Marcelo. "{CHAPTER 2 Structural Elucidation of Macromolecules}." Essential Techniques for Medical and Life Scientists: A Guide to Contemporary Methods and Current Applications with the Protocols. BENTHAM SCIENCE PUBLISHERS, 2018. 30-91. Abstract

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Bule, Pedro, Virgínia M. R. Pires, Victor D. Alves, Ana Luísa Carvalho, José A. M. Prates, Luís M. A. Ferreira, Steven P. Smith, Harry J. Gilbert, Ilit Noach, Edward A. Bayer, Shabir Najmudin, and Carlos M. G. A. Fontes. "Higher order scaffoldin assembly in Ruminococcus flavefaciens cellulosome is coordinated by a discrete cohesin-dockerin interaction." Scientific Reports. 8.1 (2018): 6987. AbstractWebsite

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.

Santarsia, Sabrina, Ana Sofia Grosso, Filipa Trovão, Jesús Jiménez-Barbero, Ana Luísa Carvalho, Cristina Nativi, and Filipa Marcelo. "Molecular recognition of a Thomsen-Friedenreich antigen mimetic targeting human galectin-3." ChemMedChem. Aug 9. doi: 10.1002/cmdc.201800525. [Epub ahead of print] (2018). AbstractWebsite

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.

Branco, Paula S., Daniela Peixoto, Margarida Figueiredo, Gabriela Malta, Catarina Roma-Rodrigues, Pedro Viana Batista, Alexandra R. Fernandes, Sónia Barroso, Ana Luisa Carvalho, Carlos A. M. Afonso, and Luísa Maria Ferreira. "Synthesis, cytotoxicity evaluation in human cell lines and in vitro DNA interaction of a hetero arylidene-9(10H)-anthrone." European Journal of Organic Chemistry (2018): n/a–n/a. AbstractWebsite

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(± 0.20) x 105 M-1) which is 10x higher than that described for doxorubicin (Kb = 3.2 (±0.23) × 104 M-1). Furthermore, compound 4 quenches the fluorescence emission of GelRed-CT-DNA system with a quenching constant (KSV) of 3.3(±0.3) x 103 M-1 calculated by the Stern-Volmer equation.

Gomes, Ana Sara, Filipa Trovão, Benedita Andrade Pinheiro, Filipe Freire, Sara Gomes, Carla Oliveira, Lucília Domingues, Maria João Romão, Lucília Saraiva, and Ana Luísa Carvalho. "The Crystal Structure of the R280K Mutant of Human p53 Explains the Loss of DNA Binding." International Journal of Molecular Sciences. 19 (2018). AbstractWebsite

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 Å. 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.

Kumar, Krishan, Márcia Correia, Virgínia R. Pires, Arun Dhillon, Kedar Sharma, Vikky Rajulapati, Carlos M. G. A. Fontes, Ana Luísa Carvalho, and Arun Goyal. "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." International Journal of Biological Macromolecules (2018): -. AbstractWebsite

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 °C. CtLam81A displayed Km, 2.1 ± 0.12 mg/ml and Vmax, 109 ± 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 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °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 Å 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.

Ribeiro, Diana O., Benedita A. Pinheiro, Ana Luisa Carvalho, and Angelina S. Palma. "Targeting protein-carbohydrate interactions in plant cell-wall biodegradation: the power of carbohydrate microarrays." Carbohydrate Chemistry: Chemical and Biological Approaches Volume 43. Vol. 43. The Royal Society of Chemistry, 2018. 159-176. 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.

2017
Kryshtafovych, Andriy, Reinhard Albrecht, Arnaud Baslé, Pedro Bule, Alessandro T. Caputo, Ana Luisa Carvalho, Kinlin L. Chao, Ron Diskin, Krzysztof Fidelis, Carlos M. G. A. Fontes, Folmer Fredslund, Harry J. Gilbert, Celia W. Goulding, Marcus D. Hartmann, Christopher S. Hayes, Osnat Herzberg, Johan C. Hill, Andrzej Joachimiak, Gert-Wieland Kohring, Roman I. Koning, Leila {Lo Leggio}, Marco Mangiagalli, Karolina Michalska, John Moult, Shabir Najmudin, Marco Nardini, Valentina Nardone, Didier Ndeh, Thanh H. Nguyen, Guido Pintacuda, Sandra Postel, Mark J. van Raaij, Pietro Roversi, Amir Shimon, Abhimanyu K. Singh, Eric J. Sundberg, Kaspars Tars, Nicole Zitzmann, and Torsten Schwede. "Target highlights from the first post-PSI CASP experiment (CASP12, May-August 2016)." Proteins: Structure, Function, and Bioinformatics (2017). AbstractWebsite

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.

Hussain, Abid, Ana T. S. Semeano, Susana I. C. J. Palma, Ana S. Pina, José Almeida, Bárbara F. Medrado, Ana C. C. S. Pádua, Ana L. Carvalho, Madalena Dionísio, Rosamaria W. C. Li, Hugo Gamboa, Rein V. Ulijn, Jonas Gruber, and Ana C. A. Roque. "Tunable gas sensing gels by cooperative assembly." Advanced Functional Materials. just accep (2017): xx. AbstractWebsite

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Pires, Virgínia M. R., Pedro M. M. Pereira, Joana L. A. Brás, Márcia Correia, Vânia Cardoso, Pedro Bule, Victor D. Alves, Shabir Najmudin, Immacolata Venditto, Luís M. A. Ferreira, Maria João Romão, Ana Luísa Carvalho, Carlos M. G. A. Fontes, and Duarte Miguel Prazeres. "Stability and ligand promiscuity of type A carbohydrate-binding modules are illustrated by the structure of Spirochaeta thermophila StCBM64C." Journal of Biological Chemistry. 292 (2017): 4847-4860. AbstractWebsite

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.

Kowacz, Magdalena, Mateusz Marchel, Lina Juknaité, José M. S. S. Esperança, Maria João Romão, Ana Luísa Carvalho, and Luís Paulo N. Rebelo. "Infrared light-induced protein crystallization. Structuring of protein interfacial water and periodic self-assembly." Journal of Crystal Growth. 457 (2017): 362-368. AbstractWebsite

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.

Bule, Pedro, Victor D. Alves, Vered Israeli-Ruimy, Ana L. Carvalho, Luís M. A. Ferreira, Steven P. Smith, Harry J. Gilbert, Shabir Najmudin, Edward A. Bayer, and Carlos M. G. A. Fontes. "Assembly of Ruminococcus flavefaciens cellulosome revealed by structures of two cohesin-dockerin complexes." Scientific Reports. 7.1 (2017): 759. AbstractWebsite

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’ 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.

Polino, Mariella, Ana Luı́sa Carvalho, Lina Juknaitė, Carla A. M. Portugal, Isabel M. Coelhoso, Maria João Romão, and João G. Crespo. "Ion-Exchange Membranes for Stable Derivatization of Protein Crystals." Crystal Growth & DesignCrystal Growth & Design (2017). AbstractWebsite
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dos Santos, Raquel, Ana Luísa Carvalho, and Cecília A. A. Roque. "Renaissance of protein crystallization and precipitation in biopharmaceuticals purification." Biotechnology Advances (2017): -. AbstractWebsite

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.

2016
Brás, Joana L. A., Benedita A. Pinheiro, Kate Cameron, Fiona Cuskin, Aldino Viegas, Shabir Najmudin, Pedro Bule, Virginia M. R. Pires, Maria João Romão, Edward A. Bayer, Holly L. Spencer, Steven Smith, Harry J. Gilbert, Victor D. Alves, Ana Luísa Carvalho, and Carlos M. G. A. Fontes. "Diverse specificity of cellulosome attachment to the bacterial cell surface." Scientific Reports. 6 (2016): 38292. AbstractWebsite

During the course of evolution, the cellulosome, one of Nature'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.

2015
Kowacz, M., M. Marchel, L. Juknaite, Jmss Esperanca, MJ Romao, AL Carvalho, and L. P. N. Rebelo. "Ionic-Liquid-Functionalized Mineral Particles for Protein Crystallization." Crystal Growth & Design. 15 (2015): 2994-3003. AbstractWebsite

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.

Palma, A. S., Y. Liu, H. Zhang, Y. Zhang, B. V. McCleary, G. Yu, Q. Huang, L. S. Guidolin, A. E. Ciocchini, A. Torosantucci, D. Wang, AL Carvalho, C. M. Fontes, B. Mulloy, R. A. Childs, T. Feizi, and W. Chai. "Unravelling glucan recognition systems by glycome microarrays using the designer approach and mass spectrometry." Mol Cell Proteomics (2015). AbstractWebsite

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.

2014
Ribeiro, Diana, Alina Kulakova, Pedro Quaresma, Eulalia Pereira, Cecilia Bonifacio, Maria Joao Romao, Ricardo Franco, and Ana Luisa Carvalho. "Use of Gold Nanoparticles as Additives in Protein Crystallization." Crystal Growth & Design. 14 (2014): 222-227. AbstractWebsite
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2013
Viegas, Aldino, Joao Sardinha, Filipe Freire, Daniel F. Duarte, Ana L. Carvalho, Carlos M. G. A. Fontes, Maria J. Romao, Anjos L. Macedo, and Eurico J. Cabrita. "Solution structure, dynamics and binding studies of a family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11)." The Biochemical journal. 451 (2013): 289-300.Website