Fluorescence spectroscopy and imaging are important biophysical techniques to study dynamics and function of proteins in vitro and in live cells. In the context of this CRC we want to perform single-molecule and ensemble fluorescence spectroscopy and multiparameter fluorescence image spectroscopy (MFIS) with high-precison Förster Resonance Energy Transfer (hpFRET) measurements to study the same proteins associated to membranes and in solution under in vitro and live cell conditions. In this way we address the problem how the change of environment during membrane association and dissociation, different chemical identities, presence of interaction partners, and local concentration influences the dynamic, structural and functional properties in prototypic proteins. We will study two prototypic protein families with a different number of domains: (1) the relatively small 14 kD protein GABARAP with one domain and (2) the large 65kD murine Guanylate Binding Proteins with three domains. The shift of the protein localization from the cytosol to the membrane is induced by a change of their identities by introducing membrane anchors such as phospholipids or isoprenyl moieties. Altogether, the suggested project of A08 must be seen in the context of an integrated approach with concerted efforts comprising different experimental techniques for in vitro and live cell studies that are complemented by simulations: (1) for GABARAP: high-resolution NMR studies by B03, cell biological studies in B02, and computer simulations in A07; (2) for mGBPs: biochemistry and cell biology in A06, computer simulations in A07, and super-resolution microscopy in Z02.
Project leader: Prof. Dr. Claus Seidel, ,
Researcher: Dr. Jakub Kubiak,
Nicolaas van der Voort M.Sc.,