Bower, J. B. | Search bioRxiv

bioRxiv2025

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). Yet this gain in baseline stability was accompanied by a paradox: LMNG, the most stabilizing detergent, supported the weakest neurotensin agonist binding affinity. Thermodynamic analysis resolved this contradiction by partitioning stability into detergent-driven conformational rigidity ({Delta}Gconf) and ligand-induced stabilization ({Delta}Gligand). In DM, {Delta}Gligand contributions were large, consistent with the receptors engineered background. In contrast, LMNG maximized {Delta}Gconf, constraining conformational flexibility and reducing {Delta}Gligand. 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. These results highlight 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. These findings emphasize that detergent choice should be guided by whether the goal is structural resolution or dynamic characterization.

biophysics↗

bioRxiv2024

Insights on the GPCR helix 8 solution structure and orientation using a neurotensin receptor 1 peptide

G-protein coupled receptors (GPCRs) are the largest class of membrane proteins in the human genome with high pharmaceutical relevance and implications to human health. These receptors share a prevalent architecture of seven transmembrane helices followed by an intracellular, amphipathic helix 8 (H8) and a disordered C-terminus. Technological advancements have led to over 1000 receptor structures in the last two decades, yet frequently H8 and the C-tail are conformationally heterogeneous or altogether absent. Here we synthesize a peptide comprising the neurotensin receptor 1 (NTS1) H8 and C-terminus (H8-Ctail) to investigate its structural stability, conformational dynamics and orientation in the presence of detergent and phospholipid micelles, which mimic the membrane. Circular dichroism (CD) and nuclear magnetic resonance (NMR) measurements confirm that zwitterionic 1,2-diheptanoyl-sn-glycero-3-phosphocholine is a potent stabilizer of H8 structure, whereas the commonly-used branched detergent lauryl maltose neopentyl glycol (LMNG) is unable to completely stabilize the helix - even at amounts four orders of magnitude greater than its critical micellar concentration. We then used NMR spectroscopy to assign the backbone chemical shifts. A series of temperature and lipid titrations were used to define the H8 boundaries as F376-R392 from chemical shift perturbations, changes in resonance intensity, and chemical-shift derived phi/psi angles. Finally, the H8 azimuthal and tilt angles, defining the helix orientation relative of the membrane normal were measured using paramagnetic relaxation enhancement (PRE) NMR. Taken together, our studies reveal the H8C-tail region is sensitive to membrane physicochemical properties and is capable of more adaptive behavior than previously suggested by static structural techniques.

biophysics↗