Genetically encoded conformational probes for small GTPase activity

Abstract

Small GTPases are guanosine nucleotide binding enzymes that exist in two states, being active in the guanosine-triphosphate- (GTP) and inactive in the guanosine-diphosphate- (GDP) bound state. Thus, these proteins act as binary molecular switches, allowing signaling exclusively in the GTP-bound state and function in a variety of cellular signaling processes, including cytoskeleton organization, membrane trafficking, nuclear transport, cell survival and proliferation. Due to their localization to different cellular compartments as well as their nucleotide- and localization-dependent activity, small GTPases are attractive targets for the development of biosensors. Recently, a new type of FRET-based sensors for small GTPase activity was developed. These conformational probes for small GTPase activity (COSGA) allow for the direct detection of conformational changes within the target protein and thereby determining the activity state of the small GTPase. The work presented in this thesis focused on the development of a second generation of COSGA probes to facilitate the preparation process and the application for cellular studies. To genetically encode the sensor design, stop codon suppression technique and intracellular fluorescence labeling were used. The first part of this work focused on the development of a genetically encoded Rab1b probe to establish the second generation sensor, whereas the aim of the second part was to apply the sensor design to Rheb. At the present time, no effector protein has been reported for Rheb, hindering the development of conventional probes to monitor Rheb activity spatiotemporally. Due to its role as an activator of mTORC1, a master regulator for cell metabolism, growth and proliferation, Rheb is a particularly interesting target. This work demonstrates the establishment of a genetically encoded Rheb sensor using the previously described conformational sensor design. This Rheb sensor allows visualization of spatiotemporal Rheb activity in living cells.