Unit of Molecular Bioenergetics
Lab of Biochemistry and Biophysics
tel. ++39 (051) 2091288
fax ++39 (051) 242576
Group Leader: Prof. G. Venturoli
E-mail : giovanni.venturoli@unibo.it
Research in progress :
- The coupling between protein dynamics and electron transfer in photosynthetic reaction centers. (F. Francia, M. Malferrari, P. Turina, G. Venturoli) The role of protein conformational dynamics in controlling key electron transfer processes within the bacterial photosynthetic reaction centers (RC) is investigated in dehydrated RC films and in RC containing glassy sugar matrices. In both systems dehydration results in a reduced conformational dynamics and strongly affects the rate of specific electron transfer reactions, mimicking at room temperature effects observed previously at cryogenic temperatures. Electron transfer kinetics are studied by visible and near-IR laser absorption spectroscopy (maximum time resolution 10^(-7) s). Water-protein interactions are examined in parallel by FTIR spectroscopy. A special interest is devoted to understand at the molecular level the RC-water-matrix dynamical coupling in glasses formed by the disaccharide trehalose. The formation of trehalose glasses upon dehydration is thought to be responsible for the surprising ability of many organisms to survive conditions of extreme drought and temperature, entering reversibly a state of anydrobiosis, which can persist for several years. The role of the RC hydration shell and of protein-water-matrix interactions in governing the RC conformational dynamics is also studied by high-field EPR spectroscopy (in collaboration with K. Moebius, Free University, Berlin, Germany) and by time-resolved differential FTIR spectroscopy (in collaboration with A. Mezzetti, Saclay, France). To gain additional information on the coupling between electron transfer and the RC dynamics we are presently extending these studies to genetically modified RCs, carrying site-specific mutations in the electron acceptor and donor regions (in collaboration with P. Sebban, University of Paris Sud, France) and A. Labahn, University of Freiburg, Germany, respectively).
- Developing innovative, dehydrated antibodies for flow cytofluorimetry based on the bioprotective properties of trehalose. (F. Francia, M. Malferrari, P. Turina, G. Venturoli) Trehalose exhibits an astonishing efficacy in protecting biomolecules against damages caused by exposure to extreme temperatures (higher than 70'C or cryogenic), as well as arising from extreme dehydration. We have recently undertaken a collaboration with Aczon S.p.A. (Bologna) aimed at exploiting these bio-protecting properties of trehalose in the development of diagnostic kits for cytofluorimetry. Co-lyophylization protocols in the presence of trehalose are currently tested, which appear to allow the long-term conservation of conjugated-antibodies in the dehydrated state at room (and higher) temperature, possibly enhancing the sensitivity of cytofluorimetric assays.
- Elucidation of the local structure of metal sites in proton translocating enzymes by X-ray Absorption Fine Structure (XAFS) spectroscopy. (F. Francia, G. Venturoli) During the last years, in collaboration with F. Boscherini (Dpt. of Physics, University of Bologna) a new synergic approach to XAFS analysis has been set up for the identification of binding motifs in protein metal sites. The approach, which can be applied even in the absence of a crystallographic structure, has been successfully used in the study of zinc binding sites in the bacterial reaction center, the cytochrome bc1 complex, the cytochrome c oxidase, the NADH-Q oxidoreductase, and the transhydrogenase. XAFS analysis of inhibitory Zn binding sites is providing valuable information to identify proton translocation pathways in redox membrane proteins. Zn binding sites of the bacterial cytochrome bc1 complex are presently investigated by coupling XAFS spectroscopy with site-specific mutagenesis (in collaboration with F. Daldal, University of Pennsylvania, Philadelphia, USA) and isothermal titration calorimetry (in collaboration with S. Ciurli, University of Bologna). XAFS spectra are acquired at the GILDA beamline of the European Synchrotron Radiation Facility (ESRF) located in Grenoble (France).
- Supramolecular organisation of the bacterial photosynthetic unit. (F. Francia, P.Turina, G. Venturoli) The structure-function relationship is studied in purified RC-antenna complexes, with emphasis on the role of the PufX protein. This protein, whose knock-out produces a non-photosynthetic phenotype, seems to induce dimerization of the RC-antenna complexes. Upon isolation, the core complex retains a functional ubiquinone pool, dispersed into a hydrophobic phase mainly constituted by cardiolipin. The different environment in which the RC is embedded (antenna plus lipids) has a strong influence on the intramolecular electron transfer reactions. Effects of the sugar matrix on the RC charge recombination kinetics, studied in this highly organized complex, could give more details on the coupling mechanism between the external matrix and the protein.
- Molecular mechanisms of energy transduction within F1FO -ATP synthase. (P. Turina) As a model system, we use both the photosynthetic bacterium Rhodobacter capsulatus and Escherichia coli. Presently we are focusing on the intrinsic uncoupling, which we have recently discovered, between ATP synthesis and proton translocation. In order to investigate its molecular mechanism and its possible physiological role, we are measuring proton fluxes across FO while monitoring ATP synthesis or hydrolysis in F1 in wild type and mutated enzymes. In collaboration with Prof. Graeber at the University of Freiburg, we investigate the relationship between the thermodynamic H+/ATP ratio and the c-to-beta-subunit ratio in different species.
Funding :
- PRIN 2008 (prot. 2008ZWHZJT) Structure-dynamics-function of biomolecules in systems far from thermodynamic ideality.
- RFO 2009 - F. Francia, P. Turina
- RFO 2008 - F. Francia, P. Turina
- RFO 2008 - G. Venturoli
- RFO 2009 - G. Venturoli
Patents :
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Selected Publications :
- Giachini, L., Veronesi, G., Francia, F., Venturoli, G. and F. Boscherini (2010) Synergic approach to XAFS analysis for the identification of most probable binding motifs for mononuclear zinc sites in metalloprotein. Journal of Synchrotron Radiation 17: 41-52.
- Veronesi, G., Whitehead, S.J., Francia, F., Giachini, L., Boscherini, F., Venturoli, G., Cotton, N.P.J. and J. Baz Jackson (2010) X-ray absorption studies of Zn2+ -binding sites in Escherichia coli transhydrogenase and its betaH91K mutant. Biochimica et Biophysica Acta 1797: 494-500.
- Francia, F., Malferrari, M., Sacquin-Mora, S. and G. Venturoli (2009) Charge recombination kinetics and protein dynamics in wild type and carotenoid-less bacterial reaction centers: studies in trehalose glasses. Journal of Physical Chemistry B 113: 10389-10398.
- Francia, F., Dezi M., Mallardi, A., Palazzo, G., Cordone, L. and G. Venturoli (2008) Protein-matrix coupling/uncoupling in 'dry' systems of photosynthetic reaction center embedded in trehalose/sucrose: the origin of trehalose peculiarity. Journal of the American Chemical Society 130: 10240-10246.
- Palazzo, G., Francia, F., Mallardi, A., Giustini, M., Lopez, F. and G. Venturoli (2008) Water activity regulates the QA- to QB electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides. Journal of the American Chemical Society 130: 9353-9363.
- D'Alessandro, M, Turina, P, Melandri, B.A. (2008) Intrinsic uncoupling in the ATP synthase of Escherichia coli. Biochim. Biophys. Acta 1777: 1518-1527.
- Steigmiller, S., Turina, P., Graeber, P. (2008) The thermodynamic H+/ATP ratios of the H+-ATPsynthases from chloroplasts and Escherichia coli. Proc. Natl. Acad. Sci. USA 105: 3745-3750.
- Veronesi, G., Giachini, L., Francia, F., Mallardi, A., Palazzo, G., Boscherini, F. and G. Venturoli (2008) The Fe2+ site of photosynthetic reaction centers probed by multiple scattering XAFS spectroscopy: improving structure resolution in dry matrices. Biophysical Journal 95: 814-822.
- Mallardi, A., Giustini, M., Lopez, F., Dezi, M., Venturoli, G. and G. Palazzo. (2007) Functionality of photosynthetic reaction centers in polyelectrolyte multilayers: toward an herbicide biosensor. Journal of Physical Chemistry B 111: 3304-3314.
- Richter, M.F., Baier, J., Prem, T., Oellerich, S., Francia, F., Venturoli, G., Oesterhelt, D., Southall, J., Cogdell, R.J. and J. Koehler (2007) Symmetry matters for the electronic structure of core complexes from Rhodopseudomonas palustris and Rhodobacter sphaeroides PufX-. Proc. Nat. Acad. Sci. USA 104: 6661-6665.
- Dezi, M., Francia, F., Mallardi, A., Colafemmina, G., Palazzo, G. and G. Venturoli (2007) Stabilization of charge separation and cardiolipin confinement in antenna-reaction center complexes purified from Rhodobacter sphaeroides. Biochimica et Biophysica Acta 1767: 1041-1056.
- Francia, F., Giachini, L., Boscherini, F., Venturoli, G., Capitanio, G., Martino, P.L., and S. Papa. (2007) The inhibitory binding site(s) of Zn2+ in cytochrome c oxidase. FEBS Letters 581: 611-616.
- Giachini, L., Francia, F., Veronesi, G., Lee, D.W., Daldal, F., Huang, L.S., Berry, E.A., Cocco, T., Papa, S., Boscherini, F. and G. Venturoli (2007) X-ray absorption studies of Zn2+ binding sites in bacterial, avian, and bovine cytochrome bc1 complexes. Biophysical Journal 93: 2934-2951.
- Giachini, L., Francia, F., Boscherini, F., Pacelli, C., Cocco, T., Papa, S. and G. Venturoli (2007) EXAFS reveals a structural zinc binding site in the bovine NADH-Q oxidoreductase. FEBS Letters 581: 5645-5648.
- Giachini, L., Francia, F., Cordone, L., Boscherini, F. and G. Venturoli (2007) Cytochrome c in a dry trehalose matrix: structural and dynamical effects probed by X-ray absorption spectroscopy. Biophysical Journal 92: 1350-1460.
- Gubellini, F., Francia, F., Turina, P., Levy, D., Venturoli, G., Melandri, B.A. (2007) Heterogeneity of photosynthetic membranes from Rhodobacter capsulatus: Size dispersion and ATP synthase distribution. Biochimica et Biophysica Acta 1767: 1340-1352.
- Gubellini, F., Francia, F., Busselez, J., Venturoli, G. and Levy, D. (2006) Functional and structural analysis of the photosynthetic apparatus of Rhodobacter veldkampii. Biochemistry 45: 10512 -10520.
- Giachini, L., Francia, F., Mallardi, A., Palazzo, G., Carpene', E., Boscherini, F. and G. Venturoli (2005) Multiple scattering X-ray absorption studies of Zn2+ binding sites in bacterial photosynthetic reaction centers. Biophysical Journal 88: 2038-2046.
- Cordone, L., Cottone, G., Giuffrida, S., Palazzo, G., Venturoli, G. and V. Viappiani (2005) Internal dynamics and protein-matrix coupling in trehalose-coated proteins. Biochimica et Biophysica Acta 1749: 252-281.
- Turina P, Giovannini D, Gubellini F, Melandri BA. (2004) Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus. Biochemistry 43: 11126-34.
- Scheuring S, Francia F, Busselez J, Melandri BA, Rigaud JL, Levy D. (2004) Structural role of PufX in the dimerization of the photosynthetic core complex of Rhodobacter sphaeroides. J Biol Chem. 279: 3620-6.
- Turina P, Samoray D, Graeber P. (2003) H+/ATP ratio of proton transport-coupled ATP synthesis and hydrolysis catalysed by CF0F1-liposomes. EMBO J. 22: 418-26.
- Francia F, Palazzo G, Mallardi A, Cordone L, Venturoli G. (2003) Residual water modulates QA- to QB electron transfer in bacterial reaction centers embedded in trehalose amorphous matrices. Biophys J 85: 2760-2775.
- Francia F, Palazzo G, Mallardi A, Cordone L, Venturoli G. (2003) Probing light-induced conformational transitions in bacterial photosynthetic reaction centers embedded in trehalose-water amorphous matrices. Biochim Biophys Acta 1658: 50-57.
- Turina P, Melandri BA. (2002) A point mutation in the ATP synthase of Rhodobacter capsulatus results in differential contributions of Delta(pH) and Delta(phi) in driving the ATP synthesis reaction. Eur J Biochem. 269: 1984-92.
- Palazzo G, Mallardi A, Hochkoeppler A, Cordone L, Venturoli G. (2002) Electron transfer kinetics in photosynthetic reaction centers embedded in trehalose glasses: trapping of conformational substates at room temperature. Biophys J. 6, n. , pp. 28-32
- Francia F, Dezi M, Rebecchi A, Mallardi A, Palazzo G, Melandri BA, Venturoli G. (2002) Light-harvesting complex 1 stabilizes P+QB- charge separation in reaction centers of Rhodobacter sphaeroides. Biochemistry 43: 14199-210.
- Turina P. (2000) Structural changes during ATP hydrolysis activity of the ATP synthase from Escherichia coli as revealed by fluorescent probes. J Bioenerg Biomembr. 32: 373-81. Review
- Francia F, Wang J, Venturoli G, Melandri BA, Barz WP, Oesterhelt D. (1999) The reaction center-LH1 antenna complex of Rhodobacter sphaeroides contains one PufX molecule which is involved in dimerization of this complex. Biochemistry 38: 6834-45.
Postgraduate training :
Ph.D. Program in Cellular, Molecular, and Industrial Biology: project n.2 - Functional and Molecular Biology
Collaborations :
- Prof. Lorenzo Cordone, Dipartimento di Scienze Fisiche ed Astronomiche, Universita' di Palermo.
- Prof. Gerardo Palazzo, Dipartimento di Chimica, Universita' di Bari.
- Prof. Federico Boscherini Dipartimento di Fisica, Universita' di Bologna.
- Prof. Dieter Oesterhelt, Department for Membrane Biochemistry, Max-Planck-Institute for Biochemistry, Martinsried, Germany.
- Dr. Daniel Levy, Institut Curie, UMR-CNRS 168 and LRC-CEA 34V, France.
- Prof. Klaus Moebius, Department of Physics, Free University Berlin, Germany.
- Prof. Wolfgang Lubitz, Dr. Anton Savitsky, Max Planck Institute for Bioinorganic Chemistry, Muelheim an der Ruhr, Germany.
- Prof. Peter Graeber, Prof Andreas Labahn, Institut fuer Physikalische Chemie, Universitaet Freiburg, Germany.
- Dr. Alfredo Ventola, Dr. Corrado Selva, Aczon SpA, Monte San Pietro, Bologna.
- Prof. Pierre Sebban, Laboratoire de Chimie Physique, Universite' Paris Sud 11, France.
- Dr. Alberto Mezzetti, Laboratoire de Spectrochimie Infrarouge et Raman, Universite' de Sciences et Technologies de Lille, France.
- Prof. Fevzi Daldal, Department of Biology, University of Pennsylvania, Philadelphia, USA.
Links :
Homepage Prof. Klaus Moebius:
http://www.physik.fu-berlin.de/forschung/emeriti/index.html
Homepage Prof. Wolfgang Lubitz:
http://ewww.mpi-muelheim.mpg.de/bac/mitarbeiter/lubitz/lubitz_en.php
Homepage Prof. Fezvi Daldal:
http://www.bio.upenn.edu/faculty/daldal/
Research group Biophysical Chemistry (Prof. P. Graeber, Prof. A. Labahn):
http://pc2.physchem.uni-freiburg.de/apache2-default/homepage/index.html
Homepage Prof. Pierre Sebban:
http://pagesperso.lcp.u-psud.fr/sebban/index.html
European Synchrotron Radiation Facility (ESFR), Grenoble:
http://www.esrf.eu/
Feher and Okamura Biophysics Lab (Bacterial Photosynthesis and Reaction Center):
http://physics.ucsd.edu/~raifeher/
A.R. Crofts Homepage (Biological Energy Conversion):
http://www.life.illinois.edu/crofts/bioph354/index.html
A.R. Crofts Homepage (bc1 complex):
http://www.life.illinois.edu/crofts/ahab/home.html
Protein Dynamics:
http://www.nature.com/nature/journal/v450/n7172/fig_tab/nature06522_F1.html
Anhydrobiosis:
http://www.nature.com/nbt/journal/v18/n2/fig_tab/nbt0200_145_F1.html
Research group :
Giovanni Venturoli (Associate Professor)
Francesco Francia (Research Associate)
M. Paola Turina (Research Associate)
Marco Malferrari (Ph.D. Student)
