Thesis supervisor: Tibor Páli
Location of studies (in Hungarian): ELKH BRC Abbreviation of location of studies: SZBK
Description of the research topic:
Continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy is a very powerful technique to measure the rotational correlation time, orientational order and lateral diffusion of spin-labeled lipids in a biomembrane. Spin-label EPR has several unique benefits in membrane-protein research: it can be used in native and reconstituted proteins and membranes; causes small or no perturbation; spectra are clean for interpretation and fitting; proteins, lipids, inhibitors, substrates all can be spin-labeled; it yields data on dynamics in an optimal time-scale for biomembranes; it can detect non-covalent interactions; and it also yields structural data. Measurement of lipid packing, fluidity and lipid-protein interaction with fluorescent and spin-labeling techniques is routine in the host lab, as well as non-linear EPR techniques developed for structural studies on membrane proteins. Therefore, the main method in this project will be state-of-the-art spin-label EPR spectroscopy, and we will use best equipped pulsed EPR instrument (in the host lab) in Central- and East-Europe. Spin-label EPR will be complemented with fluorescence spectroscopy, which will be used to measure native fluorescence of Trp amino acid (AA) in membrane proteins but also the lifetime and anisotropic rotation of fluorescent dyes in membranes. The two techniques complement each other very well for studying biomembranes: they operate in different time-scales, they have different sensitivity, the source molecular groups are free radicals or optically active and they can have different locations. Therefore, their combination on the same sample should offer new and unique benefits. Our main objective in this proposal is to implement and refine modern pulsed EPR and fluorescence spectroscopic techniques and their combination for proximity measurements in model biomembranes containing reconstituted membrane proteins. More specifically, the new methods will allow measurement of lipid-lipid, lipid-protein (lipid-AA) and protein-protein (AA-AA) proximity relations and distances in membranes. The work involves the following specific tasks:
• Model biological membranes will be measured at room temperature to study collisional interactions between EPR active and fluorescent molecules, whereas low temperature measurements will be used to study static dipolar interactions. These samples will serve as models for developing the experimental approaches.
• Selected subunits of the Vo domain of the vacuolar proton-ATPase (V-ATPase) will be spin- or fluorescence-labeled and reconstituted into model membranes. After spin-labeling the assembly of the c-d (or c-b-d) V-ATPase subunits in lipid vesicles, EPR experiments will be made to determine proximity relations. We will use modern pulsed EPR techniques for improved sensitivity and accurate distance data, possibly in collaboration with Prof. Rosa Bartucci (Department of Physics, University of Calabria). These measurements will provide detailed data on the assembly of the V-ATPase rotor and Vo domain.
• If we can detect quenching-like effect of the spin-labels on the native Trp fluorescence of subunit b, we will do time-correlated single photon counting life-time fluorescence measurements to test their proximity in the c-ring assembly.
• Computer assisted spectrum analysis and fitting algorithms will be developed in order to maximise the information on structure and dynamics from the spectra.
Required language skills: English Number of students who can be accepted: 2
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