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2014
Palma, AS, Feizi T, Childs RA, Chai W, Liu Y.  2014.  The neoglycolipid (NGL)-based oligosaccharide microarray system poised to decipher the meta-glycome. Curr Opin Chem Biol. 18:87-94. AbstractWebsite

The neoglycolipid (NGL) technology is the basis of a state-of-the-art oligosaccharide microarray system. The NGL-based microarray system in the Glycosciences Laboratory Imperial College London (http://www3.imperial.ac.uk/glycosciences) is one of the two leading platforms for glycan microarrays, being offered for screening analyses to the broad biomedical community. Highlighted in this review are the sensitivity of the analysis system and, coupled with mass spectrometry, the provision for generating 'designer' microarrays from glycomes to identify novel ligands of biological relevance. Among recent applications are assignments of ligands for apicomplexan parasites, pandemic 2009 influenza virus, polyoma and reoviruses, an innate immune receptor against fungal pathogens, Dectin-1, and a novel protein of the endoplasmic reticulum, malectin; also the characterization of an elusive cancer-associated antigen. Some other contemporary advances in glycolipid-containing arrays and microarrays are also discussed.

Pessoa, JC, Gonçalves G, Roy S, Correia I, Mehtab S, Santos MFA, Santos-Silva T.  2014.  New insights on vanadium binding to human serum transferrin. Inorganica Chimica Acta. 420:60-68. AbstractWebsite

Abstract The knowledge on the binding of vanadium ions and complexes to serum proteins and how vanadium might be transported in blood and up-taken by cells has received much attention during the last decade, particularly as far as the transport of VIVO2+ is concerned. In this work we revise and discuss some relevant aspects of previous research, namely the two main types of binding proposed for transport of VIVO(carrier)2 complexes. New results, obtained by circular dichroism (CD), \{EPR\} and gel electrophoresis, regarding the binding of vanadium to hTF in the oxidation states +5 and +3 are also presented. Namely, evidences for the binding of VV-species to diferric-transferrin, designated by (FeIII)2hTF, as well as to (AlIII)2hTF, are presented and discussed, the possibility of up-take of vanadate by cells through (FeIII)2hTF endocytosis being suggested. It is also confirmed that \{VIII\} binds strongly to hTF, forming di-vanadium(III)-transferrin, designated by (VIII)2hTF, and gel electrophoresis experiments indicate that (VIII)2hTF corresponds to a ‘closed conformation’ similar to (FeIII)2hTF.

Otrelo-Cardoso, AR, da Silva Correia MA, Schwuchow V, Svergun DI, Romao MJ, Leimkuehler S, Santos-Silva T.  2014.  Structural Data on the Periplasmic Aldehyde Oxidoreductase PaoABC from Escherichia coli: SAXS and Preliminary X-ray Crystallography Analysis. International Journal of Molecular Sciences. 15:2223-2236., Number 2 AbstractWebsite
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Ribeiro, D, Kulakova A, Quaresma P, Pereira E, Bonifacio C, Romao MJ, Franco R, Carvalho AL.  2014.  Use of Gold Nanoparticles as Additives in Protein Crystallization. Crystal Growth & Design. 14:222-227., Number 1 AbstractWebsite

Gold nanoparticles (AuNPs) exhibit unique properties that have made them a very attractive material for application in biological assays. Given the potentially interesting interactions between AuNPs and biological macromolecules, we investigated AuNPs-induced protein crystal growth. Differently functionalized AuNPs were tested as additives in cocrystallization studies with model proteins (hen egg white lysozyme (HEWL), ribonuclease A (RNase A), and proteinase K) as well as with case studies where there were problems in obtaining well-diffracting crystals. Trials were performed considering different crystallization drawbacks, from total absence of crystals to improvement of crystal morphology, size, twinning, and number of crystals per drop. Improvement of some of these factors was observed in the cases of HEWL, RNase A, phenylalanine hydroxylase (PAR), myoglobin, native aldehyde oxidase (AOH), and human albumin. In these proteins, the presence of the AuNPs promoted an increase in the size and/or better crystal morphology. From the systematic trials and subsequent observations, it can be concluded that the introduction of AuNPs should definitely be considered in crystal optimization trials to improve previously determined crystallization conditions.

Santos, MFA, Correia I, Oliveira AR, Garribba E, Pessoa JC, Santos-Silva T.  2014.  Vanadium Complexes as Prospective Therapeutics: Structural Characterization of a VIV Lysozyme Adduct. European Journal of Inorganic Chemistry. :n/a–n/a.: WILEY-VCH Verlag AbstractWebsite

The biological activity of vanadium complexes, namely, as insulin enhancers, is well known. We report a combined X-ray crystallography, electron paramagnetic resonance, and density functional theory study of the interaction of vanadium picolinate complexes with hen egg white lysozyme (HEWL). We show that the VIVO(pic)2 complex covalently binds to the COO– group of the side chain of Asp52 of HEWL. The long VIV=O bond obtained in the X-ray study is explained to be due to reduction of VIV to VIII during exposure of the crystals to the intense X-ray beam.

2013
Crusat, M, Liu J, Palma AS, Childs RA, Liu Y, Wharton SA, Lin YP, Coombs PJ, Martin SR, Matrosovich M, Chen Z, Stevens DJ, Hien VM, Thanh TT, le Nhu NT, Nguyet LA, do Ha Q, van Doorn HR, Hien TT, Conradt HS, Kiso M, Gamblin SJ, Chai W, Skehel JJ, Hay AJ, Farrar J, de Jong MD, Feizi T.  2013.  Changes in the hemagglutinin of H5N1 viruses during human infection–influence on receptor binding. Virology. 447:326-37., Number 1-2 AbstractWebsite

As avian influenza A(H5N1) viruses continue to circulate in Asia and Africa, global concerns of an imminent pandemic persist. Recent experimental studies suggest that efficient transmission between humans of current H5N1 viruses only requires a few genetic changes. An essential step is alteration of the virus hemagglutinin from preferential binding to avian receptors for the recognition of human receptors present in the upper airway. We have identified receptor-binding changes which emerged during H5N1 infection of humans, due to single amino acid substitutions, Ala134Val and Ile151Phe, in the hemagglutinin. Detailed biological, receptor-binding, and structural analyses revealed reduced binding of the mutated viruses to avian-like receptors, but without commensurate increased binding to the human-like receptors investigated, possibly reflecting a receptor-binding phenotype intermediate in adaptation to more human-like characteristics. These observations emphasize that evolution in nature of avian H5N1 viruses to efficient binding of human receptors is a complex multistep process.

Seixas, JD, Mukhopadhyay A, Santos-Silva T, Otterbein LE, Gallo DJ, Rodrigues SS, Guerreiro BH, Goncalves AML, Penacho N, Marques AR, Coelho AC, Reis PM, Romao MJ, Romao CC.  2013.  Characterization of a versatile organometallic pro-drug (CORM) for experimental CO based therapeutics. Dalton Transactions. 42:5985-5998., Number 17 AbstractWebsite
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Mukhopadhyay, A, Bursakov SA, Ramos JL, Wittich RM, Kladova AV, Romao MJ, van Dillewijn P, Carvalho AL.  2013.  Determinants of selective group reduction in the TNT-bound xenobiotic reductase B from P. putida. European Biophysics Journal with Biophysics Letters. 42:S179-S179. AbstractWebsite
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Mahro, M, Bras NF, Cerqueira NMFSA, Teutloff C, Coelho C, Romao MJ, Leimkuehler S.  2013.  Identification of Crucial Amino Acids in Mouse Aldehyde Oxidase 3 That Determine Substrate Specificity. Plos One. 8, Number 12 AbstractWebsite
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Coelho, C, Marangon J, Rodrigues D, Moura JJG, Romao MJ, Paes de Sousa PM, Correia dos Santos MM.  2013.  Induced peroxidase activity of haem containing nitrate reductases revealed by protein film electrochemistry. Journal of Electroanalytical Chemistry. 693:105-113. AbstractWebsite
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Mehtab, S, Goncalves G, Roy S, Tomaz AI, Santos-Silva T, Santos MFA, Romao MJ, Jakusch T, Kiss T, Pessoa JC.  2013.  Interaction of vanadium(IV) with human serum apo-transferrin. Journal of Inorganic Biochemistry. 121:187-195. AbstractWebsite
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Marangon, J, Correia HD, Brondino CD, Moura JJG, Romao MJ, Gonzalez PJ, Santos-Silva T.  2013.  Kinetic and Structural Studies of Aldehyde Oxidoreductase from Desulfovibrio gigas Reveal a Dithiolene-Based Chemistry for Enzyme Activation and Inhibition by H2O2. Plos One. 8, Number 12 AbstractWebsite
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Verma, AK, Goyal A, Freire F, Bule P, Venditto I, Bras JLA, Santos H, Cardoso V, Bonifacio C, Thompson A, Romao MJ, Prates JAM, Ferreira LMA, Fontes CMGA, Najmudin S.  2013.  Overexpression, crystallization and preliminary X-ray crystallographic analysis of glucuronoxylan xylanohydrolase (Xyn30A) from Clostridium thermocellum. Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 69:1440-1442. AbstractWebsite
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Viegas, A, Sardinha J, Freire F, Duarte DF, Carvalho AL, Fontes CMGA, Romao MJ, Macedo AL, Cabrita EJ.  2013.  Solution structure, dynamics and binding studies of a family 11 carbohydrate-binding module from Clostridium thermocellum (CtCBM11). Biochemical Journal. 451:289-300. AbstractWebsite
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Palma, AS, Pinheiro B, Liu Y, Takeda Y, Chai W, Ito Y, Romao MJ, Carvalho AL, Feizi T.  2013.  The Structural Basis of the Recognition of Di-glucosylated N-glycans by the ER Lectin Malectin. Glycobiology. 23:1368-1369., Number 11 AbstractWebsite
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Neu, U, Allen SA, Blaum BS, Liu Y, Frank M, Palma AS, Ströh LJ, Feizi T, Peters T, Atwood WJ, Stehle T.  2013.  A structure-guided mutation in the major capsid protein retargets BK polyomavirus. PLoS Pathog. 9:e1003688., Number 10 AbstractWebsite

Viruses within a family often vary in their cellular tropism and pathogenicity. In many cases, these variations are due to viruses switching their specificity from one cell surface receptor to another. The structural requirements that underlie such receptor switching are not well understood especially for carbohydrate-binding viruses, as methods capable of structure-specificity studies are only relatively recently being developed for carbohydrates. We have characterized the receptor specificity, structure and infectivity of the human polyomavirus BKPyV, the causative agent of polyomavirus-associated nephropathy, and uncover a molecular switch for binding different carbohydrate receptors. We show that the b-series gangliosides GD3, GD2, GD1b and GT1b all can serve as receptors for BKPyV. The crystal structure of the BKPyV capsid protein VP1 in complex with GD3 reveals contacts with two sialic acid moieties in the receptor, providing a basis for the observed specificity. Comparison with the structure of simian virus 40 (SV40) VP1 bound to ganglioside GM1 identifies the amino acid at position 68 as a determinant of specificity. Mutation of this residue from lysine in BKPyV to serine in SV40 switches the receptor specificity of BKPyV from GD3 to GM1 both in vitro and in cell culture. Our findings highlight the plasticity of viral receptor binding sites and form a template to retarget viruses to different receptors and cell types.

Neu, U, Khan ZM, Schuch B, Palma AS, Liu Y, Pawlita M, Feizi T, Stehle T.  2013.  Structures of B-lymphotropic polyomavirus VP1 in complex with oligosaccharide ligands. PLoS Pathog. 9:e1003714., Number 10 AbstractWebsite

B-Lymphotropic Polyomavirus (LPyV) serves as a paradigm of virus receptor binding and tropism, and is the closest relative of the recently discovered Human Polyomavirus 9 (HPyV9). LPyV infection depends on sialic acid on host cells, but the molecular interactions underlying LPyV-receptor binding were unknown. We find by glycan array screening that LPyV specifically recognizes a linear carbohydrate motif that contains α2,3-linked sialic acid. High-resolution crystal structures of the LPyV capsid protein VP1 alone and in complex with the trisaccharide ligands 3'-sialyllactose and 3'-sialyl-N-acetyl-lactosamine (3SL and 3SLN, respectively) show essentially identical interactions. Most contacts are contributed by the sialic acid moiety, which is almost entirely buried in a narrow, preformed cleft at the outer surface of the capsid. The recessed nature of the binding site on VP1 and the nature of the observed glycan interactions differ from those of related polyomaviruses and most other sialic acid-binding viruses, which bind sialic acid in shallow, more exposed grooves. Despite their different modes for recognition, the sialic acid binding sites of LPyV and SV40 are half-conserved, hinting at an evolutionary strategy for diversification of binding sites. Our analysis provides a structural basis for the observed specificity of LPyV for linear glycan motifs terminating in α2,3-linked sialic acid, and links the different tropisms of known LPyV strains to the receptor binding site. It also serves as a useful template for understanding the ligand-binding properties and serological crossreactivity of HPyV9.

Pinheiro, BA, Carvalho AL, Romao MJ, Fontes CM.  2013.  Study of the cohesin-dockerin interaction and its role in the C. thermocellum cellulosome assembly. European Biophysics Journal with Biophysics Letters. 42:S180-S180. AbstractWebsite
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Romao, MJ.  2013.  Unraveling new functions and modes of action of molybdenum-dependent enzymes. European Biophysics Journal with Biophysics Letters. 42:S35-S35. AbstractWebsite
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Santos, MFA, Oliveira AR, Somnath R, Romao MJ, Pessoa JC, Santos-Silva T.  2013.  Vanadium compounds as prospective therapeutics: X-ray structure of protein adducts. European Biophysics Journal with Biophysics Letters. 42:S181-S181. AbstractWebsite
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2012
Graham, LM, Gupta V, Schafer G, Reid DM, Kimberg M, Dennehy KM, Hornsell WG, Guler R, Campanero-Rhodes MA, Palma AS, Feizi T, Kim SK, Sobieszczuk P, Willment JA, Brown GD.  2012.  The C-type Lectin Receptor CLECSF8 (CLEC4D) Is Expressed by Myeloid Cells and Triggers Cellular Activation through Syk Kinase. Journal of Biological Chemistry. 287:25964-25974., Number 31 AbstractWebsite
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Palma, AS, Liu Y, Zhang Y, Zhang H, Luis AS, Carvalho AL, Gilbert HJ, Boraston A, Fontes CMGA, Chai W, Ten F.  2012.  Designer-oligosaccharide microarrays to decipher ligands in mammalian and prokaryotic glucan-recognition systems. Glycobiology. 22:1612-1613., Number 11 AbstractWebsite
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Bras, JLA, Carvalho AL, Viegas A, Najmudin S, Alves VD, Prates JAM, Ferreira LMA, Romao MJ, Gilbert HJ, Fontes CMGA.  2012.  ESCHERICHIA COLI EXPRESSION, PURIFICATION, CRYSTALLIZATION, AND STRUCTURE DETERMINATION OF BACTERIAL COHESIN-DOCKERIN COMPLEXES. Cellulases. 510(Gilbert, H. J., Ed.).:395-415. Abstract
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Coelho, C, Mahro M, Trincao J, Carvalho ATP, Ramos MJ, Terao M, Garattini E, Leimkuehler S, Romao MJ.  2012.  The First Mammalian Aldehyde Oxidase Crystal Structure INSIGHTS INTO SUBSTRATE SPECIFICITY. Journal of Biological Chemistry. 287:40690-40702., Number 48 AbstractWebsite
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Kowacz, M, Mukhopadhyay A, Carvalho AL, Esperanca J, Romao MJ, Rebelo LPN.  2012.  Hofmeister effects of ionic liquids in protein crystallization: Direct and water-mediated interactions. Crystengcomm. 14:4912-4921., Number 15 AbstractWebsite

We have performed experiments on the crystallization of two low molecular weight, positively charged proteins, lysozyme and ribonuclease A, using ionic liquids as either crystallization additives or, in particular cases, as precipitating agents. The ionic liquids (ILs) have been ordered according to their salting-in/out ability and the relative position of these ionic liquids in this ranking has been rationalized by considering their hydration properties (positive-negative, hydrophobic-hydrophilic). The ability to screen the effective charge of cationic proteins and aid protein nucleation (salting-out) has been shown to be superior for large polarizable anions with low charge density, negatively hydrated-Cl-, Br-, [SCN](-), methane-[C1SO3](-) and ethanesulfonates [C2SO3](-), than for anions with a relatively stable hydration shell, positively hydrated-lactate [Lac](-), butylsulfonate [C4SO3](-) and acetate [Ac](-). Upon increasing the background salt concentration, where electrostatic interactions are already effectively screened, the ability of the IL ions to stabilize proteins in solution (salting-in) has been shown to increase as the ions are likely to migrate to the non-polar protein surface and lower protein-water interfacial tension. This tendency is enhanced as the focus moves from those ions with positively hydrated hydrophilic compartments (e. g. [Ac](-)) to those with negatively hydrated groups (e. g. [C1SO3](-)) and the prevailing hydrophobic hydration (e. g. [C4SO3](-)). The observed inversion in the relative effect of ILs on protein crystallization with increasing ionic strength of the aqueous media has been interpreted as the differing effects of ion adsorption: charge screening and interfacial tension modification. Moreover, this work can further help in our understanding of the influence of ionic liquids on conformational changes of biomacromolecules in solution. Identification of the specific incorporation sites for choline and acetate ions, localized in two lysozyme crystals grown in pure IL solutions without any buffer or inorganic precipitant, can give us some insight into the role of the ionic liquid ions in protein structure development.