Ricardo Branco | Universidade Nova de Lisboa (original) (raw)

Papers by Ricardo Branco

Cláudia S.M. Fernandes, Ana Sofia Pina, Ana M.G.C. Dias,Ricardo J.F. Branco, Ana Cecília Afonso Roque

The green fluorescent protein (GFP) is widely employed to report on a variety of molecular phenom... more The green fluorescent protein (GFP) is widely employed to report on a variety of molecular phenomena,but its selective recovery is hampered by the lack of a low-cost and robust purification alternative. This work reports an integrated approach combining rational design and experimental validation toward theoptimization of a small fully-synthetic ligand for GFP purification. A total of 56 affinity ligands basedon a first-generation lead structure were rationally designed through molecular modeling protocols.The library of ligands was further synthesized by solid-phase combinatorial methods based on the Ugireaction and screened against Escherichia coli extracts containing GFP. Ligands A4C2, A5C5 and A5C6emerged as the new lead structures based on the high estimated theoretical affinity constants and the high GFP binding percentages and enrichment factors. The elution of GFP from these adsorbents was further characterized, where the best compromise between mild elution conditions, yiel...

PLOS ONE, 2015

Metalloproteases have evolved in a vast number of biological systems, being one of the most diver... more Metalloproteases have evolved in a vast number of biological systems, being one of the most diverse types of proteases and presenting a wide range of folds and catalytic metal ions. Given the increasing understanding of protein internal dynamics and its role in enzyme function, we are interested in assessing how the structural heterogeneity of metalloproteases translates into their dynamics. Therefore, the dynamical profile of the clan MA type protein thermolysin, derived from an Elastic Network Model of protein structure, was evaluated against those obtained from a set of experimental structures and molecular dynamics simulation trajectories. A close correspondence was obtained between modes derived from the coarse-grained model and the subspace of functionally-relevant motions observed experimentally, the later being shown to be encoded in the internal dynamics of the protein. This prompted the use of dynamics-based comparison methods that employ such coarsegrained models in a representative set of clan members, allowing for its quantitative description in terms of structural and dynamical variability. Although members show structural similarity, they nonetheless present distinct dynamical profiles, with no apparent correlation between structural and dynamical relatedness. However, previously unnoticed dynamical similarity was found between the relevant members Carboxypeptidase Pfu, Leishmanolysin, and Botulinum Neurotoxin Type A, despite sharing no structural similarity. Inspection of the respective alignments shows that dynamical similarity has a functional basis, namely the need for maintaining proper intermolecular interactions with the respective substrates. These results suggest that distinct selective pressure mechanisms act on metalloproteases at structural and dynamical levels through the course of their evolution. This work shows how new insights on metalloprotease function and evolution can be assessed with comparison schemes that incorporate information on protein dynamics. The integration of these newly developed tools, if applied to other protein families, can lead to more accurate and descriptive protein classification systems.

Frontiers in Microbiology, 2016

Patients with multiple comorbidities are often administered simultaneously or sequentially antifu... more Patients with multiple comorbidities are often administered simultaneously or sequentially antifungals and antibacterial agents, without full knowledge of the consequences of drug interactions. Considering the clinical relevance of liposomal amphotericin B (L-AMB), the association between LAMB and six antibacterial agents was evaluated against four clinical isolates and one type strain of Candida spp. and two clinical isolates and one type strain of Aspergillus fumigatus. In order to evaluate such combined effects, the minimal inhibitory concentration (MIC) of LAMB was determined in the presence of 0.5-, 1-, 2-, and 4-fold peak plasma concentrations of each of the antibacterial drugs. Since the L-AMB/colistin (CST) association was the most synergic, viability assays were performed and the physiological status induced by this association was characterized. In addition, computational molecular dynamics studies were also performed in order to clarify the molecular interaction. The maximum synergistic effect with all antibacterial agents, except CST, was reached at fourfold the usual peak plasma concentrations, resulting in 2-to 8-fold LAMB MIC reduction for Candida and 2-to 16fold for Aspergillus. For CST, the greatest synergism was registered at peak plasma concentration (3 mg/L), with 4-to 8-fold LAMB MIC reduction for Candida and 16-to 32-fold for Aspergillus. LAMB at subinhibitory concentration (0.125 mg/L) combined with CST 3 mg/L resulted in: a decrease of fungal cell viability; an increase of cell membrane permeability; an increase of cellular metabolic activity soon after 1 h of exposure, which decreased until 24 h; and an increase of ROS production up to 24 h. From the molecular dynamics studies, AMB and CST molecules shown a propensity to form a stable molecular complex in solution, conferring a recognition and binding added value for membrane intercalation. Our results demonstrate that CST interacts synergistically with LAMB , forming a stable complex, which promotes the fungicidal activity of LAMB at low concentration.

Journal of Molecular Structure-theochem, 2005

We have calculated the redox potential for a Cu, Zn superoxide dismutase active-site model. The a... more We have calculated the redox potential for a Cu, Zn superoxide dismutase active-site model. The active site of the enzyme contains a redox active cooper ion coordinated to four histidine residues. The geometry of the active site cooper complex has a characteristic distortion, whose biological significance has been discussed. The computational model contains 47 atoms representing the side chains of the histidine ligands of the copper ion. Thermodynamic parameters were calculated using density-functional methods at the B3LYP level of theory. The energies of the reduced and oxidized forms, allowed us to calculate the reduction potentials for the models. The difference between the reduction potential of a freely optimized model in water (K0.58 V) and a model mimicking the distorted active site geometry (0.84 V) corresponds to C1.42 V. It is expected that the more polar environment (water) stabilizes the oxidized state over the reduced state. However, the distortion around the cooper ion also has a fundamental role in modulating the redox potential, which can be seen by comparing the calculated redox potentials of two active site models, with unconstrained and distorted geometries, both inserted in a proteic environment (0.38 and 0.84 V, respectively). The influence of the active site distortion on the high reduction potential characteristic of this kind of copper proteins, is clearly highlighted by the results, confirming the role of the protein backbone on tuning the reduction potential that is essential for the catalytic efficiency of the enzyme. Furthermore, these preliminary results show a deep contrast with the ones that have been obtained for the Blue Copper Proteins, where it was not found any uncommon structural-function correlation between the geometry of the copper ion and its reduction potential. q

Proteins-structure Function and Bioinformatics, 2008

The molecular basis of regioselectivity of cytochrome P450 monooxygenases from Bacillus megateriu... more The molecular basis of regioselectivity of cytochrome P450 monooxygenases from Bacillus megaterium (CYP102A1) with its flexible and widely opened active site is still not well understood. In the present work (−)-α-pinene bound complexes with two triple mutants were modeled to elucidate the contribution of the three major factors that mediate selectivity: active site shape, protein flexibility, and chemical reactivity of the substrate. For the triple mutant A74G F87V L188Q (GVQ), one stable, productive conformation of the substrate (conformation I) was identified by multiple molecular dynamics simulations. The model predicts pinene epoxide as a major product (42% pinene oxide, 23% verbenol) which is in agreement with the experimental product profile (70% pinene oxide, 20% verbenol). In contrast, for the triple mutant A74G F87G L188Q (GGQ) two stable productive substrate conformations were identified (conformations IIa and IIb), and verbenol was predicted as major product (81% verbenol, 16% myrtenol), which is in agreement with experimental results (77% verbenol, 10% myrtenol). The effect of chemical reactivity of the substrate was demonstrated by comparison of (−)-α-pinene to its regioisomer (−)-β-pinene, where the product profile is shifted from 68% pinocarveol and 32% myrtanal in mutant GVQ, to 40% pinocarveol and 60% myrtanal in mutant GGQ. Our results strongly suggest a major role of residue 87 in anchoring (−)-α-pinene during substrate binding which provides a simple and elegant rationalization of the dynamic structure of this enzyme-substrate complex. Proteins 2008. © 2008 Wiley-Liss, Inc.

Copper, Zinc superoxide dismutase (CuZnSOD) catalyzes the dismutation of the toxic superoxide rad... more Copper, Zinc superoxide dismutase (CuZnSOD) catalyzes the dismutation of the toxic superoxide radical into molecular oxygen and hydrogen peroxide. Dismutation is achieved by reduction and re-oxidation of the active site copper ion, where the superoxide substrate binds. This enzyme is considered to be a perfect enzyme, as the catalytic rate is very high and diffusion controlled. The redox active copper ion is coordinated by four histidine residues in a distorted square planar geometry. Much has been written about the biological significance of the geometry distortion. It is sometimes considered that it should help to tune the redox potential of the copper ion in order to efficiently reduce the first superoxide molecule and oxidize the second one. In this work we present a series of high level theoretical calculations using realistic models, which demonstrate that the distorted geometry is fundamental for the catalytic efficiency of the enzyme by allowing substrate binding without extensive geometric reorganization of the copper complex, upon changing from four to five ligands. A lower limit for the reorganization energy is calculated here in 22 kcal/mol, which would slow down the reaction kinetics by more than 13 orders of magnitude, transforming a perfect enzyme into an inefficient one.

Cláudia S.M. Fernandes, Ana Sofia Pina, Ana M.G.C. Dias,Ricardo J.F. Branco, Ana Cecília Afonso Roque

The green fluorescent protein (GFP) is widely employed to report on a variety of molecular phenom... more The green fluorescent protein (GFP) is widely employed to report on a variety of molecular phenomena,but its selective recovery is hampered by the lack of a low-cost and robust purification alternative. This work reports an integrated approach combining rational design and experimental validation toward theoptimization of a small fully-synthetic ligand for GFP purification. A total of 56 affinity ligands basedon a first-generation lead structure were rationally designed through molecular modeling protocols.The library of ligands was further synthesized by solid-phase combinatorial methods based on the Ugireaction and screened against Escherichia coli extracts containing GFP. Ligands A4C2, A5C5 and A5C6emerged as the new lead structures based on the high estimated theoretical affinity constants and the high GFP binding percentages and enrichment factors. The elution of GFP from these adsorbents was further characterized, where the best compromise between mild elution conditions, yiel...

PLOS ONE, 2015

Metalloproteases have evolved in a vast number of biological systems, being one of the most diver... more Metalloproteases have evolved in a vast number of biological systems, being one of the most diverse types of proteases and presenting a wide range of folds and catalytic metal ions. Given the increasing understanding of protein internal dynamics and its role in enzyme function, we are interested in assessing how the structural heterogeneity of metalloproteases translates into their dynamics. Therefore, the dynamical profile of the clan MA type protein thermolysin, derived from an Elastic Network Model of protein structure, was evaluated against those obtained from a set of experimental structures and molecular dynamics simulation trajectories. A close correspondence was obtained between modes derived from the coarse-grained model and the subspace of functionally-relevant motions observed experimentally, the later being shown to be encoded in the internal dynamics of the protein. This prompted the use of dynamics-based comparison methods that employ such coarsegrained models in a representative set of clan members, allowing for its quantitative description in terms of structural and dynamical variability. Although members show structural similarity, they nonetheless present distinct dynamical profiles, with no apparent correlation between structural and dynamical relatedness. However, previously unnoticed dynamical similarity was found between the relevant members Carboxypeptidase Pfu, Leishmanolysin, and Botulinum Neurotoxin Type A, despite sharing no structural similarity. Inspection of the respective alignments shows that dynamical similarity has a functional basis, namely the need for maintaining proper intermolecular interactions with the respective substrates. These results suggest that distinct selective pressure mechanisms act on metalloproteases at structural and dynamical levels through the course of their evolution. This work shows how new insights on metalloprotease function and evolution can be assessed with comparison schemes that incorporate information on protein dynamics. The integration of these newly developed tools, if applied to other protein families, can lead to more accurate and descriptive protein classification systems.

Frontiers in Microbiology, 2016

Patients with multiple comorbidities are often administered simultaneously or sequentially antifu... more Patients with multiple comorbidities are often administered simultaneously or sequentially antifungals and antibacterial agents, without full knowledge of the consequences of drug interactions. Considering the clinical relevance of liposomal amphotericin B (L-AMB), the association between LAMB and six antibacterial agents was evaluated against four clinical isolates and one type strain of Candida spp. and two clinical isolates and one type strain of Aspergillus fumigatus. In order to evaluate such combined effects, the minimal inhibitory concentration (MIC) of LAMB was determined in the presence of 0.5-, 1-, 2-, and 4-fold peak plasma concentrations of each of the antibacterial drugs. Since the L-AMB/colistin (CST) association was the most synergic, viability assays were performed and the physiological status induced by this association was characterized. In addition, computational molecular dynamics studies were also performed in order to clarify the molecular interaction. The maximum synergistic effect with all antibacterial agents, except CST, was reached at fourfold the usual peak plasma concentrations, resulting in 2-to 8-fold LAMB MIC reduction for Candida and 2-to 16fold for Aspergillus. For CST, the greatest synergism was registered at peak plasma concentration (3 mg/L), with 4-to 8-fold LAMB MIC reduction for Candida and 16-to 32-fold for Aspergillus. LAMB at subinhibitory concentration (0.125 mg/L) combined with CST 3 mg/L resulted in: a decrease of fungal cell viability; an increase of cell membrane permeability; an increase of cellular metabolic activity soon after 1 h of exposure, which decreased until 24 h; and an increase of ROS production up to 24 h. From the molecular dynamics studies, AMB and CST molecules shown a propensity to form a stable molecular complex in solution, conferring a recognition and binding added value for membrane intercalation. Our results demonstrate that CST interacts synergistically with LAMB , forming a stable complex, which promotes the fungicidal activity of LAMB at low concentration.

Journal of Molecular Structure-theochem, 2005

We have calculated the redox potential for a Cu, Zn superoxide dismutase active-site model. The a... more We have calculated the redox potential for a Cu, Zn superoxide dismutase active-site model. The active site of the enzyme contains a redox active cooper ion coordinated to four histidine residues. The geometry of the active site cooper complex has a characteristic distortion, whose biological significance has been discussed. The computational model contains 47 atoms representing the side chains of the histidine ligands of the copper ion. Thermodynamic parameters were calculated using density-functional methods at the B3LYP level of theory. The energies of the reduced and oxidized forms, allowed us to calculate the reduction potentials for the models. The difference between the reduction potential of a freely optimized model in water (K0.58 V) and a model mimicking the distorted active site geometry (0.84 V) corresponds to C1.42 V. It is expected that the more polar environment (water) stabilizes the oxidized state over the reduced state. However, the distortion around the cooper ion also has a fundamental role in modulating the redox potential, which can be seen by comparing the calculated redox potentials of two active site models, with unconstrained and distorted geometries, both inserted in a proteic environment (0.38 and 0.84 V, respectively). The influence of the active site distortion on the high reduction potential characteristic of this kind of copper proteins, is clearly highlighted by the results, confirming the role of the protein backbone on tuning the reduction potential that is essential for the catalytic efficiency of the enzyme. Furthermore, these preliminary results show a deep contrast with the ones that have been obtained for the Blue Copper Proteins, where it was not found any uncommon structural-function correlation between the geometry of the copper ion and its reduction potential. q

Proteins-structure Function and Bioinformatics, 2008

The molecular basis of regioselectivity of cytochrome P450 monooxygenases from Bacillus megateriu... more The molecular basis of regioselectivity of cytochrome P450 monooxygenases from Bacillus megaterium (CYP102A1) with its flexible and widely opened active site is still not well understood. In the present work (−)-α-pinene bound complexes with two triple mutants were modeled to elucidate the contribution of the three major factors that mediate selectivity: active site shape, protein flexibility, and chemical reactivity of the substrate. For the triple mutant A74G F87V L188Q (GVQ), one stable, productive conformation of the substrate (conformation I) was identified by multiple molecular dynamics simulations. The model predicts pinene epoxide as a major product (42% pinene oxide, 23% verbenol) which is in agreement with the experimental product profile (70% pinene oxide, 20% verbenol). In contrast, for the triple mutant A74G F87G L188Q (GGQ) two stable productive substrate conformations were identified (conformations IIa and IIb), and verbenol was predicted as major product (81% verbenol, 16% myrtenol), which is in agreement with experimental results (77% verbenol, 10% myrtenol). The effect of chemical reactivity of the substrate was demonstrated by comparison of (−)-α-pinene to its regioisomer (−)-β-pinene, where the product profile is shifted from 68% pinocarveol and 32% myrtanal in mutant GVQ, to 40% pinocarveol and 60% myrtanal in mutant GGQ. Our results strongly suggest a major role of residue 87 in anchoring (−)-α-pinene during substrate binding which provides a simple and elegant rationalization of the dynamic structure of this enzyme-substrate complex. Proteins 2008. © 2008 Wiley-Liss, Inc.

Copper, Zinc superoxide dismutase (CuZnSOD) catalyzes the dismutation of the toxic superoxide rad... more Copper, Zinc superoxide dismutase (CuZnSOD) catalyzes the dismutation of the toxic superoxide radical into molecular oxygen and hydrogen peroxide. Dismutation is achieved by reduction and re-oxidation of the active site copper ion, where the superoxide substrate binds. This enzyme is considered to be a perfect enzyme, as the catalytic rate is very high and diffusion controlled. The redox active copper ion is coordinated by four histidine residues in a distorted square planar geometry. Much has been written about the biological significance of the geometry distortion. It is sometimes considered that it should help to tune the redox potential of the copper ion in order to efficiently reduce the first superoxide molecule and oxidize the second one. In this work we present a series of high level theoretical calculations using realistic models, which demonstrate that the distorted geometry is fundamental for the catalytic efficiency of the enzyme by allowing substrate binding without extensive geometric reorganization of the copper complex, upon changing from four to five ligands. A lower limit for the reorganization energy is calculated here in 22 kcal/mol, which would slow down the reaction kinetics by more than 13 orders of magnitude, transforming a perfect enzyme into an inefficient one.