Studying Membrane Protein-Lipid Specificity through Direct Native Mass Spectrometric Analysis from Tunable Proteoliposomes

Abstract

Native mass spectrometry (nMS) has emerged as a key analytical tool to study the organizational states of proteins and their complexes with both endogenous and exogenous ligands. Specifically, for membrane proteins, it provides a key analytical dimension to determine the identity of bound lipids and to decipher their effects on the observed structural assembly. We recently developed an approach to study membrane proteins directly from intact and tunable lipid membranes where both the biophysical properties of the membrane and its lipid compositions can be customized. Extending this, we use our liposome-nMS platform to decipher the lipid specificity of membrane proteins through their multiorganelle trafficking pathways. To demonstrate this, we used VAMP2 and reconstituted it in the endoplasmic reticulum (ER), Golgi, synaptic vesicle (SV), and plasma membrane (PM) mimicking liposomes. By directly studying VAMP2 from these customized liposomes, we show how the same transmembrane protein can bind to different sets of lipids in different organellar-mimicking membranes. Considering that the cellular trafficking pathway of most eukaryotic integral membrane proteins involves residence in multiple organellar membranes, this study highlights how the lipid-specificity of the same integral membrane protein may change depending on the membrane context. Further, leveraging the capability of the platform to study membrane proteins from liposomes with curated biophysical properties, we show how we can disentangle chemical versus biophysical properties, of individual lipids in regulating membrane protein assembly.

Figure 1

(a) nMS spectra of VAMP2 in 100%DOPC liposomes. The spectra show distinct charge state distribution from 6+ to 2+, with bound DOPC. Inset shows the negative stain EM image of the liposome.

Figure 2.

(a) nMS of VAMP2 directly from liposomes containing seven different classes of lipids at a molar ratio that mimics the class-specific composition of ER. Different lipid-bound states are marked with the respective lipids. (b) Expanded view of the 3+ charge states showing up to six lipids (PC/PI) binding. Inset shows the negative stain image of the corresponding liposome.

Figure 3.

Summary of lipid binding to VAMP2 in different organellar mimic liposomes. For each organelle, the pie chart above shows the lipid composition of the liposomes in mole%, whereas the pie chart below shows lipids that bind to VAMP2 in that organelle and their relative binding percentage. The total lipid bound VAMP2 is normalized to 100% for each organelle. The data highlights how the same membrane protein can bind to a different set of lipids, with different specificity, in different organellar membranes.

Figure 4.

UniDec derived Mass plot of semiSWEET in liposomes containing (a) 67%DOPE, 23.2%DOPG, 9.8% 18:1Cardiolipin (CL), (b) 70.9%DOPE, 27.1%DOPG, 2% 16:0–18:1DAG, and (c) 67%DOPE, 23.2%DOPG, 9.8% 18:0Dilysocardiolipin (dilyso-CL). As shown while the semiSWEET is present almost exclusively as a dimer in CL containing liposomes mimicking the gram-negative inner membrane composition, replacing CL with DAG and increasing the curvature leads to almost complete loss of the dimer. In contrast in dilyso-CL containing liposomes semiSWEET is mostly present as a dimer, highlighting the role of CL headgroup chemistry in regulating the dimeric state. The relative percentage of monomer and dimer was obtained from three independent experiment and the number on top of the peaks is mean±s.e. (n=3)

Figure 5:

nMS of different membrane proteins from detergent and proteoliposomes. (a) nMS of semiSWEET from detergent and (b) from 67%DOPE, 23.2%DOPG, 9.8% 18:1Cardiolipin proteoliposomes. (c) nMS of LacY from detergent and (d) from 67%DOPE, 23.2%DOPG, 9.8% 18:1 cardiolipin proteoliposomes. (e) nMS of AqpZ from detergent and (f) from E. coli polar lipid extract proteoliposomes. Different lipid-bound states are marked with the respective lipids. All proteoliposome samples were electrosprayed with supercharger.