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Viciosa, M. T., N. T. Correia, M. Salmeron Sanchez, AL Carvalho, MJ Romao, J. L. Gomez Ribelles, and M. Dionisio. "Real-Time Monitoring of Molecular Dynamics of Ethylene Glycol Dimethacrylate Glass Former." Journal of Physical Chemistry B. 113 (2009): 14209-14217. Abstract
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Carvalho, AL, JM Dias, L. Sanz, A. Romero, JJ Calvete, and MJ Romao. "Purification, crystallization and identification by X-ray analysis of a prostate kallikrein from horse seminal plasma." Acta Crystallographica Section D-Biological Crystallography. 57 (2001): 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.

Polino, M., H. S. Rho, M. P. Pina, R. Mallada, AL Carvalho, MJ Romão, Isabel Coelhoso, J. G. E. Gardeniers, J. G. Crespo, and Carla A. M. Portugal. "Protein Crystallization in a Microfluidic Contactor with Nafion®117 Membranes." Membranes. 11 (2021). AbstractWebsite

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’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® 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® 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’ crystals structural resolution.

Freire, Filipe, Maria Joao Romao, Anjos L. Macedo, Susana S. Aveiro, Brian J. Goodfellow, and Ana Luisa Carvalho. "Preliminary structural characterization of human SOUL, a haem-binding protein." Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 65 (2009): 723-726. Abstract
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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.

Bras, Joana L. A., Victor D. Alves, Ana Luisa Carvalho, Shabir Najmudin, Jose A. M. Prates, Luis M. A. Ferreira, David N. Bolam, Maria Joao Romao, Harry J. Gilbert, and Carlos M. G. A. Fontes. "Novel Clostridium thermocellum Type I Cohesin-Dockerin Complexes Reveal a Single Binding Mode." The Journal of biological chemistry. 287 (2012): 44394-405.Website
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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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.

Viegas, Aldino, Natercia F. Bras, Nuno M. F. S. A. Cerqueira, Pedro Alexandrino Fernandes, Jose A. M. Prates, Carlos M. G. A. Fontes, Marta Bruix, Maria Joao Romao, Ana Luisa Carvalho, Maria Joao Ramos, Anjos L. Macedo, and Eurico J. Cabrita. "Molecular determinants of ligand specificity in family 11 carbohydrate binding modules - an NMR, X-ray crystallography and computational chemistry approach." Febs Journal. 275 (2008): 2524-2535. Abstract
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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 Journal. 287 (2020): 2723-2743. AbstractWebsite

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–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.

Garcia-Alvarez, Begona, Roberto Melero, Fernando M. V. Dias, Jose A. M. Prates, Carlos M. G. A. Fontes, Steven P. Smith, Maria Joao Romao, Ana Luisa Carvalho, and Oscar Llorca. "Molecular Architecture and Structural Transitions of a Clostridium thermocellum Mini-Cellulosome." Journal of Molecular Biology. 407 (2011): 571-580. Abstract
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G., Correia Viviana, Trovão Filipa, Pinheiro Benedita A., Brás Joana L. A., Silva Lisete M., Nunes Cláudia, Coimbra Manuel A., Liu Yan, Feizi Ten, Fontes Carlos M. G. A., Mulloy Barbara, Chai Wengang, Carvalho Ana Luísa, and Palma Angelina S. "Mapping Molecular Recognition of β1,3-1,4-Glucans by a Surface Glycan-Binding Protein from the Human Gut Symbiont Bacteroides ovatus." Microbiology SpectrumMicrobiology Spectrum (2021): e01826-21. AbstractWebsite

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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

dos Santos, Raquel, Maria João Romão, Ana Cecília A. Roque, and Ana Luísa Carvalho. "Magnetic particles used in a new approach for designed protein crystallization." CrystEngComm. 23 (2021): 1083-1090. AbstractWebsite

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.

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Godinho, M. H., D. Filip, I. Costa, A. - L. Carvalho, J. L. Figueirinhas, and E. M. Terentjev. "Liquid crystalline cellulose derivative elastomer films under uniaxial strain." Cellulose. 16 (2009): 199-205. Abstract
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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.

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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Carvalho, AL, VMR Pires, TM Gloster, JP Turkenburg, JAM Prates, LMA Ferreira, MJ Romao, GJ Davies, CMGA Fontes, and HJ Gilbert. "Insights into the structural determinants of cohesin dockerin specificity revealed by the crystal structure of the type II cohesin from Clostridium thermocellum SdbA." Journal of Molecular Biology. 349 (2005): 909-915. Abstract
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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.

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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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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.

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.

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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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.

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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