Enhanced structure/function of mTSPO translocator in lipid:surfactant mixed micelles

TSPO is a ubiquitous transmembrane protein used as a pharmacological marker in neuroimaging. The only known atomic structure of mammalian TSPOs comes from the solution NMR of mouse TSPO (mTSPO) bound to the P1(11195 ligand and in a DPC surfactant environment. No structure is available in a biomimetic environment and without P1(11195 which strongly stiffens the protein. We measured the effect of different amphiphilic environments on ligand-free mTSPO to study its structure/function and find optimal solubilization conditions. By replacing the SDS surfactant, where the recombinant protein is purified, with mixed lipid:surfactant (DMPC:DPC) micelles at different ratios (0:1, 1:2, and 2:1, w:w), the a-helix content and interactions and the intrinsic tryptophan (Trp) fluorescence of mTSPO are gradually increased. Small-angle X-ray scattering (SAXS) shows a more extended mTSPO/belt complex with the addition of lipids: Dmax -95 & Aring; in DPC alone versus -142 & Aring; in DMPC:DPC (1:2). SEC-MALLS shows that the molecular composition of the mTSPO belt is -98 molecules for DPC alone and -58 DMPC and -175 DPC for DMPC:DPC (1:2). Additionally, DMPC:DPC micelles stabilize mTSPO compared to DPC alone, where the protein has a greater propensity to aggregate. These structural changes are consistent with the increased affinity of mTSPO for the P1(11195 ligand in presence of lipids (Kd -70 mM in DPC alone versus -0.91 mM in DMPC:DPC, 1:2), as measured by microscale thermophoresis (MST). In conclusion, mixed lipid:surfactant micelles open new possibilities for the stabilization of membrane proteins and for their study in solution in a more biomimetic amphiphilic environment. (c) 2024 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).