Biophysical Society Thematic Meeting | Tutzing 2026

Single-Molecule FRET: The Next 30 Years

Poster Abstracts

4-POS Board 2 A BIOPHYSICAL AND COMPUTATIONAL STUDY OF G0S2, A LIPID-DROPLET ASSOCIATED ATGL INHIBITOR Faisal Bashir 1 ; Laura Rammer 1 ; Laura Gödl 1 ; Lina Riegler-Berket 1 ; Amit Singh Sahrawat 1 ; Michael Kaltenegger 1 ; Karl Gruber 1 ; Georg Krainer 1 ; Monika Oberer 1 ; 1 University of Graz, Institute for Molecular Biosciences, Graz, Austria Lipid homeostasis is tightly regulated to maintain cellular energy balance. The small regulatory protein G0S2 (G0/G1 switch gene 2) plays a critical role in lipid metabolism through inhibition of adipose triglyceride lipase (ATGL), thereby controlling lipolysis. Lipolysis is initiated by ATGL which catalyzes triacylglycerols stored in lipid droplets, the first and rate-limiting step of triglyceride breakdown. G0S2 therefore acts as a key endogenous inhibitor of ATGL, regulating fatty acid mobilization by inhibiting its lipase activity. Structural predictions and experimental studies suggest that G0S2 lacks a classical globular fold and instead adopts a helical, partially intrinsically disordered conformation. Despite its physiological relevance, structural information on G0S2 remains limited due to its aggregation-prone nature. In this study, we aimed to characterize the structural properties of recombinant human G0S2 and validate its inhibitory activity toward ATGL. G0S2 was expressed in E. coli BL21 (DE3) cells and purified using affinity and size exclusion chromatography. The purified protein was subjected to biophysical characterization, however the size distribution analysis revealed significant aggregation. To overcome this limitation, dodecyl phosphocholine (DPC) micelles were employed to mimic a membrane-like environment and promote monodisperse state. Dynamic light scattering (DLS) confirmed a homogeneous particle distribution in the presence of DPC. Circular dichroism (CD) spectroscopy demonstrated the presence of well-defined helical conformation. We also performed molecular dynamics simulations of G0S2 in DPC micelles to evaluate its structural stability and membrane associated conformations. Functional characterization was performed using a triacylglycerol hydrolase (TGH) activity assay. G0S2 inhibited ATGL activity in a concentration-dependent manner, with significant inhibition observed at low protein concentrations. Ongoing single-molecule FRET (smFRET) experiments using engineered double-cysteine G0S2 variants aim to resolve intramolecular distances and probe conformational dynamics at the single-molecule level.

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