Effects of Detergent Micelles on Lipid Binding to Proteins in Electrospray Ionization Mass Spectrometry

Abstract

A wide variety of biological processes rely upon interactions between proteins and lipids, ranging from molecular transport to the organization of the cell membrane. It was recently established that electrospray ionization mass spectrometry (ESI-MS) is capable of capturing transient interactions between membrane proteins and their lipid environment, and a detailed understanding of the underlying processes is therefore of high importance. Here, we apply ESI-MS to investigate the factors that govern complex formation in solution and gas phases by comparing nonselective lipid binding with soluble and membrane proteins. We find that exogenously added lipids did not bind to soluble proteins, suggesting that lipids have a low propensity to form electrospray ionization adducts. The presence of detergents at increasing micelle concentrations, on the other hand, resulted in moderate lipid binding to soluble proteins. A direct ESI-MS comparison of lipid binding to the soluble protein serum albumin and to the integral membrane protein NapA shows that soluble proteins acquire fewer lipid adducts. Our results suggest that protein–lipid complexes form via contacts between proteins and mixed lipid/detergent micelles. For soluble proteins, these complexes arise from nonspecific contacts between the protein and detergent/lipid micelles in the electrospray droplet. For membrane proteins, lipids are incorporated into the surrounding micelle in solution, and complex formation occurs independently of the ESI process. We conclude that the lipids in the resulting complexes interact predominantly with sites located in the transmembrane segments, resulting in nativelike complexes that can be interrogated by MS.

Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase

“…Importantly, we find that the intact DHODH-FMN complexes are released from detergent clusters in a similar manner as integral membrane proteins ( Figure 1 C). However, the low activation energy required and the presence of detergent-free, unfolded protein suggest that the association with detergent micelles is less stable for DHODH than for integral membrane proteins, in line with its predicted peripheral membrane association ( Hanson et al., 2003 , Ilag et al., 2004 , Landreh et al., 2017 ). Taken together, our findings show that non-denaturing MS is a suitable tool to probe non-covalent interactions of peripheral membrane proteins.…”

“…The limited lipid binding indicates that the protein makes little contact with the detergent-solubilized lipids. As a result, only a few lipid molecules remain attached to the protein after detergent release, in contrast to integral membrane proteins, which bind and retain a large number of lipid adducts ( Hanson et al., 2003 , Laganowsky et al., 2014 , Landreh et al., 2017 ). We conclude that DHODH associates only superficially with membrane lipids, consistent with binding to the membrane-associated region.…”

Scratching the surface: native mass spectrometry of peripheral membrane protein complexes

“…Importantly, the proteins are thus discharged from a vehicle that essentially corresponds to their native lipid environment. In contrast, detergents can affect protein-lipid interactions [73].…”

Electrospray ionization mass spectrometric solvate cluster and multiply charged ions: a stochastic dynamic approach to 3D structural analysis

“…The determination of the solvation fee Gibbs energy (ΔG Solv ) is carried out within the framework of the assumption that the systems are at chemical equilibrium. In general, the ESI-MS has been proven as methods capable of determining the equilibrium constants of noncovalent interacting systems [100][101][102]. Figure 5 presents the functional relationships between the absolute total intensity values (I TOT ) with respect to the free Gibbs energy parameters at gas-and condenses phases.…”