Deciphering fast intra- and intermolecular dynamics of G protein-coupled receptors by Fluorescence (Cross) Correlation Spectroscopy
G-protein-coupled receptors (GPCRs) represent the most versatile and pharmacologically most important class of proteins that regulate cellular functions and constitute prominent targets for drug therapy. Although recently inactive as well as active X-ray structures have been solved, we know very little about the structural dynamics of their activation and deactivation.
This project aims to analyze the structural dynamics of GPCR activation. We will investigate the hypothesis that there are several rapid (<ms) activation steps that together constitute the activation dynamics of GPCRs beyond a two-state ‘on’-‘off’ model, and that ligands can influence these dynamics in distinct ways. We intend to resolve these rapid steps with a combined FRET-FCS approach (Fluorescence Resonance Energy Transfer and Fluorescence Correlation Spectroscopy). Structural fluctuations thereby will result in anticorrelated donor and acceptor signals due to the strong donor–acceptor distance dependence of FRET. The results are expected to provide valuable information about inter- and intramolecular dynamics of GPCR activation, notably the existence of multiple states and the kinetics of their transitions. We have developed fluorescence resonance energy transfer (FRET) techniques for GPCRs that allow the monitoring of the conformational change in GPCRs, which corresponds to agonist-induced activation. Even if it has been shown by us and others that typical activation times range between 30 and 50 ms, recent molecular modelling studies suggest movements that are about 3 orders of magnitude faster, based on microsecond binding of (low affinity) agonists to GPCRs and accommodating changes that lead to structural changes approaching the active state of GPCRs; The technique that covers the previously modelled fast dynamics as well as the already observed slower time scales is FCS. Recent approaches of FRET-FCS and technical advances such as interleaved dual-color excitation allow to ‘time-stamp’ donor and acceptor fluorescence with low background, and thus were proven to be a perfect method for studying structural fluctuations in proteins. By this FRET-FCS approach we expect to be able to distinguish kinetic components in the activation process, i.e. the potential occurrence of two or multiple intermediate activation states. In this context the FRET-FCS study, which will be related to kinetic FRET experiments from project C4, is expected to provide new insights into the dynamics time scale in GPCRs signalling.
Dr. Wagner, Julia
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