Objective The alternating access model of how membrane-embedded transport proteins translocate substrates across biological membranes has been proposed since the 1960s: membrane transporters bind substrates on one side of the membrane and release them on the other side upon conformational rearrangements in the protein. This has been experimentally confirmed by high-resolution structures of membrane transporters in different conformations and is exemplified by the conformational change in the archaeal glutamate transporter homologue GltPh from Pyrococcus horikoshii. GltPh is a trimer in which each protomer functions independently of the others. Outward and inward facing conformations suggest transport by alternating access to either side of the membrane whereby a distinct transport domain undergoes large rotational and translational movements relative to the static trimerisation domain. It is unknown how GltPh achieves this conformational rearrangement, which occurs both in the absence and presence of substrates. We propose to integrate cutting-edge techniques in membrane structural biology to identify dynamic hotspots that drive the large conformational transitions in GltPh. We will combine insights from protein crystallography with local variations of thermodynamic stability and protein dynamics measured by hydrogen/deuterium exchange to map the structural components that allow conformational change to occur. In doing so, we will obtain new insights into how GltPh functions, shedding light on the mechanism of biomedically important glutamate transporters. We will use this model system to develop strategies that allow understanding of the molecular basis of substrate transport. The methods developed would be widely applicable to other membrane transport proteins. Fields of science natural sciencesearth and related environmental sciencesgeologymineralogycrystallographynatural sciencesbiological sciencesbiochemistrybiomoleculesproteinsnatural sciencesbiological sciencesmolecular biologystructural biology Keywords glutamate transporter structure conformational dynamics crystallography thermodynamics hydrogen deuterium exchange mass spectrometry Programme(s) H2020-EU.1.3. - EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions Main Programme H2020-EU.1.3.2. - Nurturing excellence by means of cross-border and cross-sector mobility Topic(s) MSCA-IF-2014-GF - Marie Skłodowska-Curie Individual Fellowships (IF-GF) Call for proposal H2020-MSCA-IF-2014 See other projects for this call Funding Scheme MSCA-IF - Marie Skłodowska-Curie Individual Fellowships (IF) Coordinator CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS Net EU contribution € 246 668,40 Address RUE MICHEL ANGE 3 75794 Paris France See on map Region Ile-de-France Ile-de-France Paris Activity type Research Organisations Links Contact the organisation Opens in new window Website Opens in new window Participation in EU R&I programmes Opens in new window HORIZON collaboration network Opens in new window Total cost € 246 668,40 Partners (1) Sort alphabetically Sort by Net EU contribution Expand all Collapse all Partner Partner organisations contribute to the implementation of the action, but do not sign the Grant Agreement. CORNELL UNIVERSITY United States Net EU contribution € 0,00 Address PINE TREE ROAD 373 14850 Ithaca Ny See on map Activity type Higher or Secondary Education Establishments Links Contact the organisation Opens in new window Website Opens in new window Participation in EU R&I programmes Opens in new window HORIZON collaboration network Opens in new window Total cost € 160 130,40