Project 11: Single-molecule studies of secondary-active transporters and their environmental dependence

In this project, we will determine conformational states and distributions of GAT1 and its structural homologues to understand how structure is related to transport. The entire functional cycle will be studied with different single-molecule approaches on timescales ranging from microseconds of seconds. The methods include fluorescence microscopy, single-molecule detection schemes via confocal and TIRF microscopy, and multi-parameter fluorescence spectroscopy. We expect to achieve a detailed description of all essential steps within the transport cycle for different biochemical conditions and lipid compositions. In the project we will collaborate with Christine Ziegler (UREG) to develop protocols for transporter purification and reconstitution and we will work with Fraser MacMillan (UEA) to obtain complementary EPR measurements.

The main objectives are:

• to use single-molecule methods such as fluorescence quenching, anisotropy and FRET to resolve all relevant conformations of the GABA transporter GAT1 and that of structural homologues, using reconstituted proteins
• to study influence of biochemical conditions such as ion and substrate concentrations, inhibitors or functional mutations in the transporter
• to determine structures and conformational ensembles of unresolved, but regulatory important regions in GAT1 by combining smFRET with ab initio structure prediction methods in detergent environment, reconstituted GAT1 in nanodiscs- and proteoliposomes
• to study the effect of the signalling lipid PIP2 and of regulatory proteins on the conformational dynamics; this will be complemented by a systematic study on the effect of distinct lipid compositions on transport and regulation
• to carry out multi-parameter fluorescence studies and compare the results with others from the network to determine the set of exchanging conformers and establish their sequence in the transport cycle