Sulfo-DIBMA encapsulation uniquely preserves signalling-competent active states of the class B1 GPCRs, calcitonin gene-related peptide and parathyroid hormone 1 receptors, in native-like nanodiscs

Class B1 G-protein-coupled receptors (GPCRs), such as the calcitonin gene-related peptide (CGRP) receptor and parathyroid hormone 1 (PTH ₁ ) receptor, require native lipid interactions to maintain signalling-competent conformations. However, conventional detergents disrupt these environments. Amphipathic copolymers offer a detergent-free alternative, yet the field still lacks a clear understanding of which polymer architectures best preserve active-state GPCR pharmacology, limiting their broader translational utility. Here, we examine how distinct copolymer chemistries influence the functional integrity of class B1 GPCRs by comparing SMA 2000, DIBMA-12, and the electroneutral sulfo-DIBMA.

Using NanoLuciferase bioluminescence resonance energy transfer (NanoBRET) ligand-binding, competition, and mini-G-protein recruitment assays on nanodisc-encapsulated receptors, we show that all three copolymers maintain high-affinity extracellular ligand binding but differ markedly in their ability to preserve intracellular signalling. Despite lower receptor extraction efficiency, only sulfo-DIBMA support mini-Gαs engagement at the CGRP receptor and enable G-protein-dependent allosteric modulation at the PTH ₁ receptor, including conserved ligand affinity and prolonged residence time. These data reveal that polymer charge and backbone chemistry, rather than extraction yield, determine whether native-like nanodiscs retain the conformational landscape required for active-state signalling. Controlling non-specific ligand binding to the copolymer is a key requirement for a successful assay.

Our findings identify sulfo-DIBMALP as a particularly superior environment for preserving native signalling behaviour in class B1 GPCRs, highlighting copolymer chemistry as an important determinant in detergent-free membrane protein studies.

HIGHLIGHTS

• Sulfo-DIBMA encapsulated nanodiscs preserve active-state conformation of human calcitonin gene-related peptide receptor and parathyroid hormone 1 receptor.

• All three copolymers (SMA 2000, DIBMA-12 and sulfo-DIBMA) preserve extracellular ligand binding but only sulfo-DIBMA preserves intracellular functional competence, including mini-Gαs recruitment and G-protein-dependent allosteric modulation.

• Copolymer chemistry, particularly the electroneutral, aliphatic nature of sulfo-DIBMA, may influence the preservation of signalling-competent states in two class B1 GPCRs by minimising charge-driven perturbations during solubilisation.

• Sulfo-DIBMALP provides a novel platform for studying dynamic membrane proteins with potential to provide mechanistic insights and facilitate drug discovery programmes in the future.

GRAPHICAL ABSTRACT