Membrane proteins play critical biochemical roles but remain challenging to study. Recently, native or nondenaturing mass spectrometry (MS) has made great strides in characterizing membrane protein interactions. However, conventional native MS relies on detergent micelles, which may disrupt natural interactions. Lipoprotein nanodiscs provide a platform to present membrane proteins for native MS within a lipid bilayer environment, but prior native MS of membrane proteins in nanodiscs has been limited by the intermediate stability of nanodiscs. It is difficult to eject membrane proteins from nanodiscs for native MS but also difficult to retain intact nanodisc complexes with membrane proteins inside. Here, we employed chemical reagents that modulate the charge acquired during electrospray ionization (ESI). By modulating ESI conditions, we could either eject the membrane protein complex with few bound lipids or capture the intact membrane protein nanodisc complex-allowing measurement of membrane protein oligomeric state within an intact lipid bilayer environment. The dramatic differences in the stability of nanodiscs under different ESI conditions opens new applications for native MS of nanodiscs.

Lipoprotein nanodiscs are ideally suited for native mass spectrometry because they provide a relatively monodisperse nanoscale lipid bilayer environment for delivering membrane proteins into the gas phase. However, native mass spectrometry of nanodiscs produces complex spectra that can be challenging to assign unambiguously. To simplify interpretation of nanodisc spectra, we engineered a series of mutant membrane scaffold proteins (MSP) that do not affect nanodisc formation but shift the masses of nanodiscs in a controllable way, eliminating isobaric interference from the lipids. Moreover, by mixing two different belts before assembly, the stoichiometry of MSP is encoded in the peak shape, which allows the stoichiometry to be assigned unambiguously from a single spectrum. Finally, we demonstrate the use of mixed belt nanodiscs with embedded membrane proteins to confirm the dissociation of MSP prior to desolvation.

Membrane proteins can be incorporated into the lipidic cubic phase (LCP) for crystal growth and structure determination. LCP crystallization has become an important tool in the field of membrane protein crystallography (particularly, but not solely, with GPCRs and other small membrane proteins). However, many details of this process are not well understood. There is little direct experimental evidence for the localization of protein and detergent after incorporation into LCP; the mechanisms of nucleation and crystal growth; and the details of how the cubic phase modifies the interactions between protein molecules. We are using Small Angle Neutron and X-Ray Scattering (SANS/SAXS) to study each step of the cubic phase crystallization process using Bacteriorhodopsin (bR) as a model system. Using SANS, it is possible to contrast-match the non-protein components of the system, i.e. detergents and lipids. This allows us to measure the protein scattering directly and in isolation, greatly simplifying the data interpretation from these complex multicomponent systems. At high bR concentrations, it is possible to measure structure factors, from which information on protein-protein interactions can be obtained. We have measured the concentration-dependent scattering of bR: (1) in solution;(2) after incorporation into LCP; and (3) as a function of precipitant concentration. Solution structure factor measurements at lower salt concentrations are consistent with a charged sphere interaction model. In contrastmatched LCP at lower concentrations of bR and precipitant, scattering from bR monomers could be observed, similarly to bR in solution. At higher bR and precipitant concentrations, a series of higher-order structures were observed by SANS, as well as protein-dependent Bragg reflections in samples in which macroscopic crystals were later observed.