Carmo, H, Gomes D, Carvalho F, Remiao F, Branco P, Ferreira L, de Pinho PG, de Bastos ML.
2009.
Hyponatremic effect caused by 3,4-methylenedioxymethamphetamine (ecstasy) in rats: The role of metabolic bioactivation, SEP 13. TOXICOLOGY LETTERS. 189:S110-S111.: European Soc Toxicol
Abstractn/a
Almeida, RM, Pauleta SR, Moura I, Moura JJ.
2009.
Rubredoxin as a paramagnetic relaxation-inducing probe, Sep. J Inorg Biochem. 103:1245-53., Number 9
AbstractThe paramagnetic effect due to the presence of a metal center with unpaired electrons is no longer considered a hindrance in protein NMR spectroscopy. In the present work, the paramagnetic effect due to the presence of a metal center with unpaired electrons was used to map the interface of an electron transfer complex. Desulfovibrio gigas cytochrome c(3) was chosen as target to study the effect of the paramagnetic probe, Fe-rubredoxin, which produced specific line broadening in the heme IV methyl resonances M2(1) and M18(1). The rubredoxin binding surface in the complex with cytochrome c(3) was identified in a heteronuclear 2D NMR titration. The identified heme methyls on cytochrome c(3) are involved in the binding interface of the complex, a result that is in agreement with the predicted complexes obtained by restrained molecular docking, which shows a cluster of possible solutions near heme IV. The use of a paramagnetic probe in (1)HNMR titration and the mapping of the complex interface, in combination with a molecular simulation algorithm proved to be a valuable strategy to study electron transfer complexes involving non-heme iron proteins and cytochromes.
Gonzalez, PJ, Barrera GI, Rizzi AC, Moura JJ, Passeggi MC, Brondino CD.
2009.
EPR studies of the Mo-enzyme aldehyde oxidoreductase from Desulfovibrio gigas: an application of the Bloch-Wangsness-Redfield theory to a system containing weakly-coupled paramagnetic redox centers with different relaxation rates, Oct. J Inorg Biochem. 103:1342-6., Number 10
AbstractElectron transfer proteins and redox enzymes containing paramagnetic redox centers with different relaxation rates are widespread in nature. Despite both the long distances and chemical paths connecting these centers, they can present weak magnetic couplings produced by spin-spin interactions such as dipolar and isotropic exchange. We present here a theoretical model based on the Bloch-Wangsness-Redfield theory to analyze the dependence with temperature of EPR spectra of interacting pairs of spin 1/2 centers having different relaxation rates, as is the case of the molybdenum-containing enzyme aldehyde oxidoreductase from Desulfovibrio gigas. We analyze the changes of the EPR spectra of the slow relaxing center (Mo(V)) induced by the faster relaxing center (FeS center). At high temperatures, when the relaxation time T(1) of the fast relaxing center is very short, the magnetic coupling between centers is averaged to zero. Conversely, at low temperatures when T(1) is longer, no modulation of the coupling between metal centers can be detected.
Rivas, MG, Mota CS, Pauleta SR, Carepo MS, Folgosa F, Andrade SL, Fauque G, Pereira AS, Tavares P, Calvete JJ, Moura I, Moura JJ.
2009.
Isolation and characterization of a new Cu-Fe protein from Desulfovibrio aminophilus DSM12254, Oct. J Inorg Biochem. 103:1314-22., Number 10
AbstractThe isolation and characterization of a new metalloprotein containing Cu and Fe atoms is reported. The as-isolated Cu-Fe protein shows an UV-visible spectrum with absorption bands at 320 nm, 409 nm and 615 nm. Molecular mass of the native protein along with denaturating electrophoresis and mass spectrometry data show that this protein is a multimer consisting of 14+/-1 subunits of 15254.3+/-7.6 Da. Mossbauer spectroscopy data of the as-isolated Cu-Fe protein is consistent with the presence of [2Fe-2S](2+) centers. Data interpretation of the dithionite reduced protein suggest that the metallic cluster could be constituted by two ferromagnetically coupled [2Fe-2S](+) spin delocalized pairs. The biochemical properties of the Cu-Fe protein are similar to the recently reported molybdenum resistance associated protein from Desulfovibrio, D. alaskensis. Furthermore, a BLAST search from the DNA deduced amino acid sequence shows that the Cu-Fe protein has homology with proteins annotated as zinc resistance associated proteins from Desulfovibrio, D. alaskensis, D. vulgaris Hildenborough, D. piger ATCC 29098. These facts suggest a possible role of the Cu-Fe protein in metal tolerance.
Rivas, MG, Mota CS, Pauleta SR, Carepo MSP, Folgosa F, Andrade SLA, Fauque G, Pereira AS, Tavares P, Calvete JJ, Moura I, Moura JJG.
2009.
Isolation and characterization of a new Cu-Fe protein from Desulfovibrio aminophilus DSM12254, Oct. Journal Of Inorganic Biochemistry. {103}:{1314-1322}., Number {10, SI}
AbstractThe isolation and characterization of a new metalloprotein containing Cu and Fe atoms is reported. The as-isolated Cu-Fe protein shows an UV-visible spectrum with absorption bands at 320 nm, 409 nm and 615 nm. Molecular mass of the native protein along with denaturating electrophoresis and mass spectrometry data show that this protein is a multimer consisting of 14 +/- 1 subunits of 15254.3 +/- 7.6 Da. Mossbauer spectroscopy data of the as-isolated Cu-Fe protein is consistent with the presence of [2Fe-2S](2+) centers. Data interpretation of the dithionite reduced protein suggest that the metallic cluster could be constituted by two ferromagnetically coupled [2Fe-2S](+) spin delocalized pairs. The biochemical properties of the Cu-Fe protein are similar to the recently reported molybdenum resistance associated protein from Desulfovibrio, D. alaskensis. Further-more, a BLAST search from the DNA deduced amino acid sequence shows that the Cu-Fe protein has homology with proteins annotated as zinc resistance associated proteins from Desulfovibrio, D. alaskensis, D. vulgaris Hildenborough, D. piger ATCC 29098. These facts suggest a possible role of the Cu-Fe protein in metal tolerance. (C) 2009 Published by Elsevier Inc.
Xie, X, Hadt RG, Pauleta SR, Gonzalez PJ, Un S, Moura I, Solomon EI.
2009.
A variable temperature spectroscopic study on Paracoccus pantotrophus pseudoazurin: Protein constraints on the blue Cu site, Oct. Journal of Inorganic Biochemistry. 103:1307-1313., Number 10
AbstractThe blue or Type 1 (T1) copper site of Paracoccus pantotrophus pseudoazurin exhibits significant absorption intensity in both the 450 and 600 nm regions. These are sigma and pi S(Cys) to Cu(2+) charge transfer (CT) transitions. The temperature dependent absorption, EPR, and resonance Raman (rR) vibrations enhanced by these bands indicate that a single species is present at all temperatures. This contrasts the temperature dependent behavior of the T1 center in nitrite reductase [S. Ghosh, X. Xie, A. Dey, Y. Sun, C. Scholes, E. Solomon, Proc. Natl. Acad. Sci. 106 (2009) 4969-4974] which has a thioether ligand that is unconstrained by the protein. The lack of temperature dependence in the T1 site in pseudoazurin indicates the presence of a protein constraint similar to the blue Cu site in plastocyanin where the thioether ligand is constrained at 2.8 angstrom. However, plastocyanin exhibits only pi CT. This spectral difference between pseudoazurin and plastocyanin reflects a coupled distortion of the site where the axial thiorether in pseudoazurin is also constrained, but at a shorter Cu-S(Met) bond length. This leads to an increase in the Cu(2+)-S(Cys) bond length, and the site undergoes a partial tetragonal distortion in pseudoazurin. Thus, its ground state wavefunction has both sigma and pi character in the Cu(2+)-S(Cys) bond. (C) 2009 Elsevier Inc. All rights reserved.
Conrath, K, Pereira AS, Martins CE, Timoteo CG, Tavares P, Spinelli S, Kinne J, Flaudrops C, Cambillau C, Muyldermans S, Moura I, Moura JJ, Tegoni M, Desmyter A.
2009.
Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductase, Mar. Protein Sci. 18:619-28., Number 3
AbstractNitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N(2)O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.
Moniz, AB, Paulos MR.
2009.
{The clothing industry as a globalized sector: implications for work organisation, quality of work and job content}, Jun. , Number 13/2009: Universidade Nova de Lisboa, IET/CICS.NOVA-Interdisciplinary Centre on Social Sciences, Faculty of Science and Technology
AbstractThe clothing sector in several countries is still seen, in many aspects as a traditional sector with some average characteristics, nevertheless is a very important sector in terms of labour market. Globalization and de-localization are having a strong impact in the organisation of work and in occupational careers. Very few companies are able to keep a position in the market without changes in organisation of work and workers, founding different ways to face this reality according to size, capital and position. We could find two main paths: one where companies outsource production to another territory, close and/or dismissal the workers; other path, where companies up skilled their capacities. This paper will present some results from the European project WORKS – Work organisation and restructuring in the knowledge society (6th Framework Programme), focusing the Portuguese case studies in several clothing companies in a comparative analysis with some other European countries.
Najmudin, S, Bonifacio C, Duarte AG, Pauleta SR, Moura I, Moura JJ, Romao MJ.
2009.
Crystallization and crystallographic analysis of the apo form of the orange protein (ORP) from Desulfovibrio gigas, Jul 1. Acta Crystallogr Sect F Struct Biol Cryst Commun. 65:730-2., Number Pt 7
AbstractThe orange-coloured protein (ORP) from Desulfovibrio gigas is a 12 kDa protein that contains a novel mixed-metal sulfide cluster of the type [S(2)MoS(2)CuS(2)MoS(2)]. Diffracting crystals of the apo form of ORP have been obtained. Data have been collected for the apo form of ORP to 2.25 A resolution in-house and to beyond 2.0 A resolution at ESRF, Grenoble. The crystals belonged to a trigonal space group, with unit-cell parameters a = 43, b = 43, c = 106 A.
Rivas, MG, Carepo MS, Mota CS, Korbas M, Durand MC, Lopes AT, Brondino CD, Pereira AS, George GN, Dolla A, Moura JJ, Moura I.
2009.
Molybdenum induces the expression of a protein containing a new heterometallic Mo-Fe cluster in Desulfovibrio alaskensis, Feb 10. Biochemistry. 48:873-82., Number 5
AbstractThe characterization of a novel Mo-Fe protein (MorP) associated with a system that responds to Mo in Desulfovibrio alaskensis is reported. Biochemical characterization shows that MorP is a periplasmic homomultimer of high molecular weight (260 +/- 13 kDa) consisting of 16-18 monomers of 15321.1 +/- 0.5 Da. The UV/visible absorption spectrum of the as-isolated protein shows absorption peaks around 280, 320, and 570 nm with extinction coefficients of 18700, 12800, and 5000 M(-1) cm(-1), respectively. Metal content, EXAFS data and DFT calculations support the presence of a Mo-2S-[2Fe-2S]-2S-Mo cluster never reported before. Analysis of the available genomes from Desulfovibrio species shows that the MorP encoding gene is located downstream of a sensor and a regulator gene. This type of gene arrangement, called two component system, is used by the cell to regulate diverse physiological processes in response to changes in environmental conditions. Increase of both gene expression and protein production was observed when cells were cultured in the presence of 45 microM molybdenum. Involvement of this system in Mo tolerance of sulfate reducing bacteria is proposed.
Paulos, MR, Moniz AB.
2009.
Are societal changes new? Questions or trends and future perceptions on knowledge-based economy, Feb , Number 02/2009: Universidade Nova de Lisboa, IET-Research on Enterprise and Work Innovation, Faculty of Science and Technology
AbstractWith the emergence of a global division of labour, the internationalisation of markets and cultures, the growing power of supranational organisations and the spread of new information technologies to every field of life, it starts to appear a different kind of society, different from the industrial society, and called by many as ‘the knowledge-based economy’, emphasizing the importance of information and knowledge in many areas of work and organisation of societies. Despite the common trends of evolution, these transformations do not necessarily produce a convergence of national and regional social and economic structures, but a diversity of realities emerging from the relations between economic and political context on one hand and the companies and their strategies on the other. In this sense, which future can we expect to the knowledge economy? How can we measure it and why is it important? This paper will present some results from the European project WORKS – Work organisation and restructuring in the knowledge society (6th Framework Programme), focusing the future visions and possible future trends in different countries, sectors and industries, given empirical evidences of the case studies applied in several European countries, underling the importance of foresight exercises to design policies, prevent uncontrolled risks and anticipate alternatives, leading to different ‘knowledge economies’ and not to the ‘knowledge economy’.
Paulos, MR, Moniz AB.
2009.
{Are societal changes new? Questions or trends and future perceptions on knowledge-based economy}, Feb , Number 02/2009: Universidade Nova de Lisboa, IET/CICS.NOVA-Interdisciplinary Centre on Social Sciences, Faculty of Science and Technology
AbstractWith the emergence of a global division of labour, the internationalisation of markets and cultures, the growing power of supranational organisations and the spread of new information technologies to every field of life, it starts to appear a different kind of society, different from the industrial society, and called by many as ‘the knowledge-based economy’, emphasizing the importance of information and knowledge in many areas of work and organisation of societies. Despite the common trends of evolution, these transformations do not necessarily produce a convergence of national and regional social and economic structures, but a diversity of realities emerging from the relations between economic and political context on one hand and the companies and their strategies on the other. In this sense, which future can we expect to the knowledge economy? How can we measure it and why is it important? This paper will present some results from the European project WORKS – Work organisation and restructuring in the knowledge society (6th Framework Programme), focusing the future visions and possible future trends in different countries, sectors and industries, given empirical evidences of the case studies applied in several European countries, underling the importance of foresight exercises to design policies, prevent uncontrolled risks and anticipate alternatives, leading to different ‘knowledge economies’ and not to the ‘knowledge economy’.
Giestas, L, Petrov V, Baptista {PV}, Lima {JC}.
2009.
General FRET-based coding for application in multiplexing methods, aug. Photochemical & Photobiological Sciences. 8:1130–1138., Number 8: Springer
AbstractFRET can be used as a strategy to assign different simultaneous events in the same sample but {"}cross-talk{"} problems are a limitation. Here we present a contribution for the better understanding of the {"}cross-talk{"} in FRET experiments that include several pairs in the same sample. Using oligonucleotide probes labeled with fluorescent dyes which can be selectively excited at a specific wavelength, and using target oligonucleotides tagged with a fluorescent dye with specific characteristics that allow only it to emit light upon selective excitation of a specific probe by energy transfer (FRET), we aim to identify the exact probe-target hybridized pair. When using three donors to probe the presence of complementary targets, only 20% of possible donor/acceptor combinations give straightforward signals readily identifiable with the sample composition, while in the remaining cases severe cross-excitation prevents the direct identification of the sample composition. To correctly resolve the samples identity, we developed a theoretical model that enables the unequivocal attribution of a sample composition to a given set of fluorescence signals, in the presence of three donors.
Conrath, K, Pereira AS, Martins CE, Timoteo CG, Tavares P, Spinelli S, Kinne J, Flaudrops C, Cambillau C, Muyldermans S, Moura I, Moura JJG, Tegoni M, Desmyter A.
2009.
Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductase, Apr. PROTEIN SCIENCE. {18}:{619-628}., Number {3}
AbstractNitric Oxide Reductase (NOR) is an integral membrane protein performing the reduction of NO to N(2)O. NOR is composed of two subunits: the large one (NorB) is a bundle of 12 transmembrane helices (TMH). It contains a b type heme and a binuclear iron site, which is believed to be the catalytic site, comprising a heme b and a non-hemic iron. The small subunit (NorC) harbors a cytochrome c and is attached to the membrane through a unique TMH. With the aim to perform structural and functional studies of NOR, we have immunized dromedaries with NOR and produced several antibody fragments of the heavy chain (VHHs, also known as nanobodies (TM)). These fragments have been used to develop a faster NOR purification procedure, to proceed to crystallization assays and to analyze the electron transfer of electron donors. BIAcore experiments have revealed that up to three VHHs can bind concomitantly to NOR with affinities in the nanomolar range. This is the first example of the use of VHHs with an integral membrane protein. Our results indicate that VHHs are able to recognize with high affinity distinct epitopes on this class of proteins, and can be used as versatile and valuable tool for purification, functional study and crystallization of integral membrane proteins.
Pina, AS, Roque ACA.
2009.
Studies on the molecular recognition between bioactive peptides and angiotensin-converting enzyme, apr. Journal of Molecular Recognition. 22:162–168., Number 2
AbstractHigh blood pressure or hypertension is a condition affecting many individuals and represents a controllable risk factor for cardiovascular diseases such as coronary heart disease and stroke. A non-pharmacological approach to manage these includes the application of food components with antihypertensive activity. Milk protein-derived peptides have been exploited as natural hypotensive agents, namely the peptides {Val-Pro-Pro} {(VPP)} and {Ile-Pro-Pro} {(IPP)}, already commercialized in functional foods as a potential alternative to synthetic drugs. These bioactive peptides inhibit in vitro and in vivo the Angiotensin I-converting enzyme {(ACE)}, a protein with an important role in blood pressure regulation. In this work, we attempted to elucidate the possible mode of interaction between the peptides and {ACE}, including mechanisms of binding to the cofactor Zn2+, and further contrast this with the known mode of inhibition exerted by synthetic drugs {(Captopril}, Enalaprilat and Lisinopril). The bioactive peptide {Ala-Leu-Pro-Met-His-Ile-Arg} {(ALPMHIR)}, also known to inhibit the enzyme {ACE} but with a lower efficiency than {VPP} and {IPP}, was utilized in the docking studies for comparison. It was observed that the best docking poses obtained for {VPP} and {IPP} were located at the {ACE} catalytic site with very high resemblance to the drugs mode of interaction, including the coordination with Zn2+. As for {ALPMHIR}, the best docking poses were located in the narrow {ACE} channel outside the catalytic site, representing higher affinity energies and fewer resemblances with the interaction established by drugs.