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.

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.

Interactions between membrane proteins and lipids are often crucial for structure and function yet difficult to define because of their dynamic and heterogeneous nature. Here, we use mass spectrometry to demonstrate that membrane protein oligomers ejected from nanodiscs in the gas phase retain large numbers of lipid interactions. The complex mass spectra that result from gas‐phase dissociation were assigned using a Bayesian deconvolution algorithm together with mass defect analysis, allowing us to count individual lipid molecules bound to membrane proteins. Comparison of the lipid distributions measured by mass spectrometry with molecular dynamics simulations reveals that the distributions correspond to distinct lipid shells that vary according to the type of protein–lipid interactions. Our results demonstrate that nanodiscs offer the potential for native mass spectrometry to probe interactions between membrane proteins and the wider lipid environment.

ConspectusMembrane proteins play critical physiological roles and make up the majority of drug targets. Due to their generally low expression levels and amphipathic nature, membrane proteins represent challenging molecular entities for biophysical study. Mass spectrometry offers several sensitive approaches to study the biophysics of membrane proteins.By preserving noncovalent interactions in the gas phase and using collisional activation to remove solubilization agents inside the mass spectrometer, native mass spectrometry (MS) is capable of studying isolated assemblies that would be insoluble in aqueous solution, such as membrane protein oligomers and protein-lipid complexes. Conventional methods use detergent to solubilize the protein prior to electrospray ionization. Gas-phase activation inside the mass spectrometer removes the detergent to yield the isolated proteins with bound ligands. This approach has proven highly successful for ionizing membrane proteins. With the appropriate choice of detergents, membrane proteins with bound lipid species can be observed, which allows characterization of protein-lipid interactions. However, detergents have several limitations. They do not necessarily replicate the native lipid bilayer environment, and only a small number of protein-lipid interactions can be resolved.In this Account, we summarize the development of different membrane mimetics as cassettes for MS analysis of membrane proteins. Examples include amphipols, bicelles, and picodiscs with a special emphasis on lipoprotein Nanodiscs. Polydispersity and heterogeneity of the membrane mimetic cassette is a critical issue for study by MS. Ever more complex datasets consisting of overlapping protein charge states and multiple lipid-bound entities have required development of new computational, theoretical, and experimental approaches to interpret both mass and ion mobility spectra. We will present the rationale and limitations of these approaches.Starting with the early work on empty Nanodiscs, we chart developments that culminate in recent high-resolution studies of membrane protein-lipid complexes ejected from Nanodiscs. For the latter, increasing collision energies allowed progressive removal of Nanodisc components, beginning with the scaffold proteins and continuing through successive shells of lipids, allowing direct characterization of the stoichiometry of the annular lipid belt that surrounds the membrane protein. We consider future directions for the study of membrane proteins in membrane mimetics, including the development of mixed lipid systems and native bilayer environments. Unambiguous assignment of these heterogeneous systems will rely heavily upon further enhancements in both data analysis protocols and instrumental resolution. We anticipate that these developments will provide new insights into the factors that control dynamic protein-lipid interactions in a variety of tailored and natural lipid environments. Detergents have driven membrane protein MSLipid membranes are a critical component of cellu...

Mass spectrometry (MS) has emerged as a powerful tool to study membrane protein complexes and protein-lipid interactions. Because they provide a precisely-defined lipid bilayer environment, lipoprotein Nanodiscs offer a promising cassette for membrane protein MS analysis. However, heterogeneous lipids create several potential challenges for native MS: additional spectral complexity, ambiguous assignments, and differing gas-phase behaviors. Here, we present strategies to address these challenges and streamline analysis of heterogeneous-lipid Nanodiscs. We show that using two lipids of similar mass limits the complexity of the spectra in heterogeneous Nanodiscs and that the lipid composition can be determined by using a dual Fourier transform approach to obtain the average lipid mass. Further, the relationship between gas-phase behavior, lipid composition, and instrumental polarity was investigated to determine the effects of lipid head group chemistry on Nanodisc dissociation mechanisms. These results provide unique mechanistic and methodological insights into characterization of complex and heterogeneous systems by mass spectrometry.