Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

10-POS Board 6 LIPIDS MODULATE CONFORMATIONAL DYNAMICS OF THE GLUTAMATE TRANSPORTER EAAT1 Qianyi Wu 1 ; Rupasree Brahma 1,2 ; Olga Boudker 1,2 ; 1 Weill Cornell Medicine, Department of Biochemistry and Biophysics, New York, NY, USA 2 Howard Hughes Medical Institute, New York, NY, USA Excitatory amino acid transporter 1 (EAAT1) clears synaptic glutamate via an elevator-like transport mechanism, in which its transport domain carries substrate and coupled ions across the membrane as it cycles through outward-facing, intermediate, and inward-facing conformations. High-resolution structures have established the molecular mechanism of transport, yet how the surrounding lipid environment shapes the conformational energy landscape and state-to-state transition rates remains poorly understood. Here, we use single-molecule Förster resonance energy transfer (smFRET) to quantify state occupancies, state transition timing, and dynamic heterogeneity of EAAT1 in membranes of defined lipid composition. Purified, site-specifically labeled EAAT1 is reconstituted into liposomes containing defined synthetic lipid mixtures. Preliminary smFRET measurements demonstrate that a membrane environment is required for EAAT1 conformational dynamics, which are abolished in detergent micelles. Cholesterol hemisuccinate (CHS), a soluble cholesterol analog, suppresses transitions, indicating that the lipid environment regulates EAAT1 dynamics. Building on these observations, we are investigating the roles of individual lipid classes and native-like membrane compositions. These dynamic measurements are complemented by functional single-transporter smFRET activity assays. Together, this integrated approach will elucidate how membrane composition governs the conformational landscape and function of EAAT1.

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