Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer

Abstract

Hierarchical organization of integral membrane proteins (IMP) and lipids at the membrane is essential for regulating myriad downstream signaling. A quantitative understanding of these processes requires both detections of oligomeric organization of IMPs and lipids directly from intact membranes and determination of key membrane components/properties that regulate them. Addressing this, we have developed a platform that enables native mass spectrometry (nMS) analysis of IMP-lipid complexes directly from intact and customizable lipid membranes. Both the lipid composition and membrane properties (such as curvature, tension, fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteo-lipid vesicles can yield the oligomeric states of the embedded IMPs, identify bound lipids, and determine the membrane properties that can regulate the observed IMP-lipid organization. Applying this, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter.

Native mass spectrometry of proteoliposomes containing integral and peripheral membrane proteins

“…Unlike G b1g2 and encapsulated soluble proteins, integral membrane proteins required the addition of m-NBA, a result consistent with previous reports. 29,30 Other supercharging agents, such as sulfolane, 63 had a similar effect.…”

“…30 Similar to nanodiscs, the addition of super charging molecules aids the ejection of membrane protein complexes from these liposomes and the ejected complexes retain bound lipids. 29,30 This approach has been particularly useful to study how the lipid environment inuences the monomer-dimer equilibrium of semisweet. 30 The integration of integral and peripheral membrane proteins with liposomes of dened composition is an attractive route for functional assays, 36,37 cryoEM studies, [38][39][40] and MS analysis of MAPs.…”

To 200,000 m/z and beyond: native electron capture charge reduction mass spectrometry deconvolves heterogeneous signals in large biopharmaceutical analytes

“…Electric fields are used to control ions in the mass spectrometer, and in combination with a background collision gas, ions can be accelerated to cause collisional energy transfer, for example to achieve fragmentation, dissociation or desolvation; the extent of acceleration and therefore activation at a given voltage is related to the ion's charge, and charge manipulation can therefore help to control the extent of activation. Supercharging has for example been shown to assist in desolvation for some membrane proteins, for which greater activation is needed to eject the protein from detergent micelles; however, in other cases, charge reduction is useful to prevent unfolding of the protein due to over-activation or internal Coulomb repulsion, and is also of fundamental interest for understanding protein structure in the gas phase (12,(14)(15)(16)(17)(18).…”

Mobile barrier mechanisms for Na+-coupled symport in an MFS sugar transporter

“…Salmonella enterica serovar Typhimurium melibiose permease (MelBSt) is the prototype Na + /solute transporter of the major facilitator superfamilies (MFS) 11,[15][16][17][18][19][20][21][22][23][24] . It catalyzes the stoichiometric symport of galactopyranoside with Na + , H + , or Li +25 .…”