Stabilization versus flexibility: Detergent-dependent trade-offs in neurotensin receptor 1 GPCR ensembles

Detergents provide essential membrane-mimetic environments for studying G protein-coupled receptors (GPCRs), but their molecular impact on receptor energetics remains incompletely understood. We combined ligand binding, thermostability measurements, and atomistic molecular dynamics to dissect detergent- versus ligand-driven stabilization in a thermostabilized neurotensin receptor 1 (enNTS1). Circular dichroism and ligand binding assays revealed that apo enNTS1 becomes progressively more stable in decyl maltoside (DM), dodecyl maltoside (DDM), and lauryl maltose neopentyl glycol (LMNG). However, this gain in baseline stability was accompanied by an initially counterintuitive observation: LMNG, the most stabilizing detergent, supported the weakest neurotensin agonist binding affinity. Thermodynamic analysis shows that this behavior arises naturally from partitioning stability between detergent-driven conformational rigidity (ΔG_conf) and ligand-induced stabilization (ΔG_ligand). In DM, ΔG_ligand contributions were large, consistent with the receptor's engineered background. In contrast, LMNG maximized ΔG_conf, constraining conformational flexibility and reducing ΔG_ligand. Molecular dynamics simulations corroborated these results, showing that LMNG formed denser, less mobile detergent shells around the receptor, enhancing protein-detergent interaction energies while limiting conformational flexibility. Redistribution of ligand contacts, particularly at neurotensin residue Y11, further underscored detergent-dependent modulation of the binding pocket. The results in this thermostabilized neurotensin receptor illustrate a fundamental trade-off: LMNG provides exceptional receptor stabilization, supporting structural studies, but may mask conformational states relevant to signaling. In contrast, less rigid detergents preserve ligand-induced transitions at the expense of stability. We therefore propose this system as a case study of how detergent chemistry can redistribute stability between conformational rigidity and ligand-induced effects, with implications for guiding detergent choice depending on whether the goal is structural resolution or dynamic characterization.