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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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Fernandes, AR, Mendonça-Martins I, Santos MFA, Raposo LR, Mendes R, Marques J, Romão CC, Romão MJ, Santos-Silva T, Baptista PV.  2020.  Improving the Anti-inflammatory Response via Gold Nanoparticle Vectorization of CO-Releasing Molecules, 2020. ACS Biomaterials Science & EngineeringACS Biomaterials Science & Engineering. 6(2):1090-1101.: American Chemical Society AbstractWebsite

CO-releasing molecules (CORMs) have been widely studied for their anti-inflammatory, antiapoptotic, and antiproliferative effects. CORM-3 is a water-soluble Ru-based metal carbonyl complex, which metallates serum proteins and readily releases CO in biological media. In this work, we evaluated the anti-inflammatory and wound-healing effects of gold nanoparticles–CORM-3 conjugates, AuNPs@PEG@BSA·Ru(CO)x, exploring its use as an efficient CO carrier. Our results suggest that the nanoformulation was capable of inducing a more pronounced cell effect, at the anti-inflammatory level and a faster tissue repair, probably derived from a rapid cell uptake of the nanoformulation that results in the increase of CO inside the cell.CO-releasing molecules (CORMs) have been widely studied for their anti-inflammatory, antiapoptotic, and antiproliferative effects. CORM-3 is a water-soluble Ru-based metal carbonyl complex, which metallates serum proteins and readily releases CO in biological media. In this work, we evaluated the anti-inflammatory and wound-healing effects of gold nanoparticles–CORM-3 conjugates, AuNPs@PEG@BSA·Ru(CO)x, exploring its use as an efficient CO carrier. Our results suggest that the nanoformulation was capable of inducing a more pronounced cell effect, at the anti-inflammatory level and a faster tissue repair, probably derived from a rapid cell uptake of the nanoformulation that results in the increase of CO inside the cell.

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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Kowacz, M, Marchel M, Juknaité L, Esperança JMSS, Romão MJ, Carvalho AL, Rebelo LPN.  2017.  Infrared light-induced protein crystallization. Structuring of protein interfacial water and periodic self-assembly. Journal of Crystal Growth. 457:362-368. AbstractWebsite

Abstract We show that a physical trigger, a non-ionizing infrared (IR) radiation at wavelengths strongly absorbed by liquid water, can be used to induce and kinetically control protein (periodic) self-assembly in solution. This phenomenon is explained by considering the effect of İR\} light on the structuring of protein interfacial water. Our results indicate that the İR\} 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 İR\} light) can prevent the protein non-specific aggregation favoring periodic self-assembly. Recognizing the ability to affect protein-water interactions by means of İR\} radiation may have important implications for biological and bio-inspired systems.

Carvalho, AL, Pires VMR, Gloster TM, Turkenburg JP, Prates JAM, Ferreira LMA, Romao MJ, Davies GJ, Fontes C, Gilbert HJ.  2005.  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. 349:909-915., Number 5 AbstractWebsite
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Cerqueira, NMFSA, Coelho C, Bras NF, Fernandes PA, Garattini E, Terao M, Romao MJ, Ramos MJ.  2015.  Insights into the structural determinants of substrate specificity and activity in mouse aldehyde oxidases. Journal of Biological Inorganic Chemistry. 20:209-217., Number 2 AbstractWebsite

In this work, a combination of homology modeling and molecular dynamics (MD) simulations was used to investigate the factors that modulate substrate specificity and activity of the mouse AOX isoforms: mAOX1, mAOX2 (previously mAOX3l1), mAOX3 and mAOX4. The results indicate that the AOX isoform structures are highly preserved and even more conserved than the corresponding amino acid sequences. The only differences are at the protein surface and substrate-binding site region. The substrate-binding site of all isoforms consists of two regions: the active site, which is highly conserved among all isoforms, and a isoform-specific region located above. We predict that mAOX1 accepts a broader range of substrates of different shape, size and nature relative to the other isoforms. In contrast, mAOX4 appears to accept a more restricted range of substrates. Its narrow and hydrophobic binding site indicates that it only accepts small hydrophobic substrates. Although mAOX2 and mAOX3 are very similar to each other, we propose the following pairs of overlapping substrate specificities: mAOX2/mAOX4 and mAOX3/mAXO1. Based on these considerations, we propose that the catalytic activity between all isoforms should be similar but the differences observed in the binding site might influence the substrate specificity of each enzyme. These results also suggest that the presence of several AOX isoforms in mouse allows them to oxidize more efficiently a wider range of substrates. This contrasts with the same or other organisms that only express one isoform and are less efficient or incapable of oxidizing the same type of substrates.

Gomes, D, Correia MAS, Romão MJ, Passarinha LA, Sousa A.  2023.  Integrated approaches for the separation and purification of recombinant HPV16 E6 protein from Escherichia coli crude extracts, 2023. 315:123647. AbstractWebsite

Human papillomavirus (HPV) is a sexually transmissible virus responsible for 5% of global human cancers and associated with 99% of cervical cancer cases. The oncogenic potential of high-risk HPVs is mainly related to the E6 and E7 oncoproteins, which are responsible, at least in part, for inactivating the p53 and pRb tumor suppressor proteins. Due to the critical role of the E6 protein in malignant tumorigenesis, it is widely recognized as a therapeutic target for anti-HPV drug development. Nevertheless, it is required to obtain large amounts of protein with high purity to perform biointeraction studies with the potential inhibitor drugs. In this work, recombinant dual-tagged E6 protein (His6-MBP-E6) was expressed from Escherichia coli (E. coli) cultures and successfully extracted by sonication/ice cycles. Affinity chromatography using MBPtrap columns allowed 85 ± 5% protein recovery with the elimination of major host heterologous proteins in a single fraction. Subsequently, a polishing step was studied by applying anionic exchange (QSepharose), size exclusion (Superdex), or immobilized-metal affinity chromatography (HisTrap). The combination of affinity chromatography with size exclusion or two affinity chromatography techniques allowed us to obtain 82 ± 2% and 94 ± 3%, of highly pure His6-MBP-E6, respectively. Also, the secondary structure of His6-MBP-E6 is preserved in both purification strategies, as appraised by circular dichroism and western-blot studies. Thermal shift assay confirmed the CD results and suggested potential additives for protein stabilization. Altogether, the reproducible strategies established for the purification of His6-MBP-E6 protein could be successfully applied to later perform biointeraction studies and structural characterization of protein–ligand complexes.

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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Mota, C, Diniz A, Coelho C, Santos-Silva T, Esmaeeli M, Leimkühler S, Cabrita EJ, Marcelo F, Romão MJ.  2021.  Interrogating the Inhibition Mechanisms of Human Aldehyde Oxidase by X-ray Crystallography and NMR Spectroscopy: The Raloxifene Case, 2021. Journal of Medicinal ChemistryJournal of Medicinal Chemistry. : American Chemical Society AbstractWebsite

Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors—thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug–drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors—thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug–drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.

Vidinha, P, Lourenco NMT, Pinheiro C, Bras AR, Carvalho T, Santos-Silva T, Mukhopadhyay A, Romao MJ, Parola J, Dionisio M, Cabral JMS, Afonso CAM, Barreiros S.  2008.  Ion jelly: a tailor-made conducting material for smart electrochemical devices. Chemical Communications. :5842-5844., Number 44 AbstractWebsite
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Polino, M, Carvalho AL, Juknaitė L, Portugal CAM, Coelhoso IM, Romão MJ, Crespo JG.  2017.  Ion-Exchange Membranes for Stable Derivatization of Protein Crystals, 2017. Crystal Growth & DesignCrystal Growth & Design. : American Chemical Society AbstractWebsite
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Sadeghi, SJ, Valetti F, Cunha CA, Romao MJ, Soares CM, Gilardi G.  2000.  Ionic strength dependence of the non-physiological electron transfer between flavodoxin and cytochrome c(553) from D-vulgaris. Journal of Biological Inorganic Chemistry. 5:730-737., Number 6 AbstractWebsite
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Kowacz, M, Marchel M, Juknaite L, Esperanca J, Romao MJ, Carvalho AL, Rebelo LPN.  2015.  Ionic-Liquid-Functionalized Mineral Particles for Protein Crystallization. Crystal Growth & Design. 15:2994-3003., Number 6 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.

Almeida, MG, Macieira S, Goncalves LL, Huber R, Cunha CA, Romao MJ, Costa C, Lampreia J, Moura JJG, Moura I.  2003.  The isolation and characterization of cytochrome c nitrite reductase subunits (NrfA and NrfH) from Desulfovibrio desulfuricans ATCC 27774 - Re-evaluation of the spectroscopic data and redox properties. European Journal of Biochemistry. 270:3904-3915., Number 19 AbstractWebsite
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