G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets. Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P binding. Endogenous lipids were also observed bound directly to the trimeric Gαβγ protein complex of the adenosine A receptor (AR) in the gas phase. Using engineered Gα subunits (mini-Gα mini-Gα and mini-Gα), we demonstrate that the complex of mini-Gα with the β adrenergic receptor (βAR) is stabilized by the binding of two PtdIns(4,5)P molecules. By contrast, PtdIns(4,5)P does not stabilize coupling between βAR and other Gα subunits (mini-Gα or mini-Gα) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P. Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gαβγ complex in the presence of PtdIns(4,5)P provide further evidence for a specific effect of PtdIns(4,5)P on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs.

Small molecules are known to stabilise membrane proteins and to modulate function and oligomeric state, but their identity is often hard to define. Here we develop and apply a high-resolution, Orbitrap mass spectrometer for intact membrane protein-ligand complexes. Using this platform we resolve the complexity of multiple binding events, quantify small molecule binding and reveal selectivity for endogenous lipids that differ only in acyl chain length.

Detergents enable the purification of membrane proteins and are indispensable reagents in structural biology. Even though a large variety of detergents have been developed in the last century, the challenge remains to identify guidelines that allow fine-tuning of detergents for individual applications in membrane protein research. Addressing this challenge, here we introduce the family of oligoglycerol detergents (OGDs). Native mass spectrometry (MS) reveals that the modular OGD architecture offers the ability to control protein purification and to preserve interactions with native membrane lipids during purification. In addition to a broad range of bacterial membrane proteins, OGDs also enable the purification and analysis of a functional G-protein coupled receptor (GPCR). Moreover, given the modular design of these detergents, we anticipate fine-tuning of their properties for specific applications in structural biology. Seen from a broader perspective, this represents a significant advance for the investigation of membrane proteins and their interactions with lipids.

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The effects of protein–ligand interactions on protein stability are typically monitored by a number of established solution-phase assays. Few translate readily to membrane proteins. We have developed an ion-mobility mass spectrometry approach, which discerns ligand binding to both soluble and membrane proteins directly via both changes in mass and ion mobility, and assesses the effects of these interactions on protein stability through measuring resistance to unfolding. Protein unfolding is induced through collisional activation, which causes changes in protein structure and consequently gas-phase mobility. This enables detailed characterization of the ligand-binding effects on the protein with unprecedented sensitivity. Here we describe the method and software required to extract from ion mobility data the parameters that enable a quantitative analysis of individual binding events. This methodology holds great promise for investigating biologically significant interactions between membrane proteins and both drugs and lipids that are recalcitrant to characterization by other means.