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Book Chapter
Ribeiro, D. O., F. Bonnardel, A. S. Palma, A. L. M. Carvalho, and S. Perez. "CBMcarb-DB: interface of the three-dimensional landscape of carbohydrate-binding modules." Carbohydrate Chemistry: Chemical and Biological Approaches Volume 46. Eds. Amélia Pilar Rauter, Yves Queneau, and Angelina Sá Palma. Vol. 46. Royal Society of Chemistry, 2024. 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–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–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.

Bras, Joana L. A., Ana Luisa Carvalho, Aldino Viegas, Shabir Najmudin, Victor D. Alves, Jose A. M. Prates, Luis M. A. Ferreira, Maria J. Romao, Harry J. Gilbert, and Carlos M. G. A. Fontes. "Escherichia coli expression, purification, crystallization, and structure determination of bacterial cohesin-dockerin complexes." Methods in enzymology. Vol. 510. 2012. 395-415. 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.

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.

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.

Carvalho, Ana Luísa, José Trincão, and Maria João Romão. "X-Ray Crystallography in Drug Discovery." Methods in molecular biology (Clifton, N.J.). Vol. 572. 2010. 31-56. 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.

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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Journal Article
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.

Carvalho, AL, FMV Dias, JAM Prates, T. Nagy, HJ Gilbert, GJ Davies, LMA Ferreira, MJ Romao, and CMGA Fontes. "Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex." Proceedings of the National Academy of Sciences of the United States of America. 100 (2003): 13809-13814. Abstract
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Carvalho, AL, L. Sanz, D. Barettino, A. Romero, JJ Calvete, and MJ Romao. "Crystal structure of a prostate kallikrein isolated from stallion seminal plasma: A homologue of human PSA." Journal of Molecular Biology. 322 (2002): 325-337. Abstract
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Romao, MJ, I. Kolln, JM Dias, AL Carvalho, A. Romero, P. F. Varela, L. Sanz, E. Topfer-Petersen, and JJ Calvete. "Crystal structure of acidic seminal fluid protein (aSFP) at 1.9 angstrom resolution: a bovine polypeptide of the spermadhesin family." Journal of Molecular Biology. 274 (1997): 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.

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.

Romero, A., MJ Romao, P. F. Varela, I. Kolln, JM Dias, AL Carvalho, L. Sanz, E. TopferPetersen, and JJ Calvete. "The crystal structures of two spermadhesins reveal the CUB domain fold." Nature Structural Biology. 4 (1997): 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.

Dias, JM, AL Carvalho, I. Kolln, JJ Calvete, E. TopferPetersen, P. F. Varela, A. Romero, C. Urbanke, and MJ Romao. "Crystallization and preliminary x-ray diffraction studies of aSFP, a bovine seminal plasma protein with a single CUB domain architecture." Protein Science. 6 (1997): 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

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.

Duarte, Marlene, Aldino Viegas, Victor D. Alves, José A. M. Prates, Luís M. A. Ferreira, Shabir Najmudin, Eurico J. Cabrita, Ana Luísa Carvalho, Carlos M. G. A. Fontes, and Pedro Bule. "A dual cohesin–dockerin complex binding mode in Bacteroides cellulosolvens contributes to the size and complexity of its cellulosome." Journal of Biological Chemistry. 296 (2021): 100552. AbstractWebsite

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–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–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.

Silva, José Malanho, Linda Cerofolini, Ana Luísa Carvalho, Enrico Ravera, Marco Fragai, Giacomo Parigi, Anjos L. Macedo, Carlos F. G. C. Geraldes, and Claudio Luchinat. "Elucidating the concentration-dependent effects of thiocyanate binding to carbonic anhydrase." 244 (2023): 112222. AbstractWebsite

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.

Carvalho, Ana Luisa, Fernando M. V. Dias, Tibor Nagy, Jose A. M. Prates, Mark R. Proctor, Nicola Smith, Edward A. Bayer, Gideon J. Davies, Luis M. A. Ferreira, Maria J. Romao, Carlos M. G. A. Fontes, and Harry J. Gilbert. "Evidence for a dual binding mode of dockerin modules to cohesins." Proceedings of the National Academy of Sciences of the United States of America. 104 (2007): 3089-3094. Abstract
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Carvalho, AL, A. Goyal, JAM Prates, DN Bolam, HJ Gilbert, VMR Pires, LMA Ferreira, A. Planas, MJ Romao, and CMGA Fontes. "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 of Biological Chemistry. 279 (2004): 34785-34793. Abstract
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Caseiro, Catarina, Nicholas G. S. McGregor, Victor Diogo Alves, Ana Luísa Carvalho, Maria João Romão, Gideon J. Davies, Carlos M. G. A. Fontes, and Pedro Bule. "Family GH157 enzyme exhibits broad linkage tolerance and a dual endo/exo- β -glucanase activity on β-glucans." (2024): 137402. AbstractWebsite

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 °C and 48 °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-1,3–1,6 activity constitutes an expansion on our understanding of β-glucan deconstruction, with the potential to inspire future applications.

Santos-Silva, T., J. Trincao, AL Carvalho, C. Bonifacio, F. Auchere, P. Raleiras, I. Moura, JJG Moura, and MJ Romao. "The first crystal structure of class III superoxide reductase from Treponema pallidum." Journal of Biological Inorganic Chemistry. 11 (2006): 548-558. Abstract
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Pinheiro, B. A., J. L. A. Bras, S. Najmudin, AL Carvalho, LMA Ferreira, JAM Prates, and CMGA Fontes. "Flexibility and specificity of the cohesin-dockerin interaction: implications for cellulosome assembly and functionality." Biocatalysis and Biotransformation. 30 (2012): 309-315. AbstractWebsite

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.

Coelho, Catarina, Pablo J. Gonzalez, Jose Trincao, Ana L. Carvalho, Shabir Najmudin, Thomas Hettman, Stephan Dieckman, Jose J. G. Moura, Isabel Moura, and Maria J. Romao. "Heterodimeric nitrate reductase (NapAB) from Cupriavidus necator H16: purification, crystallization and preliminary X-ray analysis." Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 63 (2007): 516-519. Abstract
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Dias, Ana Margarida Gonçalves Carvalho, Inês Pimentel Moreira, Iana Lychko, Cátia Lopes Soares, Arianna Nurrito, Arménio Jorge Moura Barbosa, Viviane Lutz-Bueno, Raffaele Mezzenga, Ana Luísa Carvalho, Ana Sofia Pina, and Ana Cecília Afonso Roque. "Hierarchical self-assembly of a reflectin-derived peptide." Frontiers in Chemistry. 11 (2023). AbstractWebsite

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’s basic unit—the protopeptide sequence YMDMSGYQ—as a means to understand reflectin’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.

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.

Kowacz, Magdalena, Abhik Mukhopadhyay, Ana Luisa Carvalho, Jose M. S. S. Esperanca, Maria J. Romao, and Luis Paulo N. Rebelo. "Hofmeister effects of ionic liquids in protein crystallization: Direct and water-mediated interactions." Crystengcomm. 14 (2012): 4912-4921. AbstractWebsite
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