Objective
The principal objective of this proposal is to carry out a comprehensive program in structural biology to understand the mechanism of proton pumping by membrane bound proton pumps. The first work package aims at a determination of the three-dimensional atomic structure of bacteriorhodopsin(bR), one of natures light driven proton pumps, recorded at different time points in the photo cycle. The intermediate states in the photo cycle will be recorded using single crystals of bR combined with millisecond to picosecond diffraction techniques. The combination of high quality crystals, two photon reaction initiation and short x-ray pulses should make it possible to record the structure of the K. L and M intermediates at high resolution which is expected to pinpoint the proton translocation pathway. Complimentary experiments will exploit the temperature dependence of the photo cycle and isolate the intermediates by freeze trapping. Moreover crystals of bR mutants with longer lived intermediates will also be studied.
In the second work package we will examine chemically driven proton pumps by looking at oxygen reduction in the haem-containing respiratory oxidases quinol oxidase, cytochrome oxidase, and cytochrome cdl. These enzymes diffract to high resolution and are active in the crystal. Fast diffraction methods combined with spectroscopic techniques will be used to correlate electronic transitions with structural transitions. These techniques will be used to produce a three-dimensional movie of oxygen reduction by respiratory oxidases. The experiments are expected to reveal the oxygen chemistry in cytochrome c oxidase, which will aid our understanding of proton pumping in such enzymes. The studies will allow us to see how redox energy can be switched into conformational energy within a haem protein, a phenomenon of general importance.
The third work package deals with the optimization of stroposcopic diffraction techniques at the European Synchrotron Radiation Facility. It is foreseen to prepare the ID09 beamline for micro crystal diffraction, upgrade the x-ray source with a mini-gap undulator with 170 poles, install a high-flux Bragg-Bragg monochromator and to mount a femto-second microphotospectrometer on the sample goniostat. These developments will enable us to run stroposcopic diffraction experiments on the 50 picosecond, nanosecond, microsecond and milli-sec timescale and have on-line diagnostics of the excitation characteristics of the sample.
Fields of science (EuroSciVoc)
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CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques.
- humanitiesartsmodern and contemporary artcinematography
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes
- natural sciencesbiological sciencesmolecular biologystructural biology
- natural sciencesphysical sciencestheoretical physicsparticle physicsphotons
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Call for proposal
Data not availableFunding Scheme
CSC - Cost-sharing contractsCoordinator
38043 GRENOBLE
France