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 and 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, and fluidity) of these bilayers can be precisely customized to a target membrane. Subsequent direct nMS analysis of these intact proteolipid 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 method, we show how lipid binding regulates neurotransmitter release and how membrane composition regulates the functional oligomeric state of a transporter.
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Data availability
Data for the main figures and extended data figures are available from figshare: https://doi.org/10.6084/m9.figshare.21788915.v4Source data are provided with this paper.
References
Simons, K. & Toomre, D. Lipid rafts and signal transduction. Nat. Rev. Mol. Cell Biol. 1, 31–39 (2000).
Andersen, O. S. & Koeppe, R. E. Bilayer thickness and membrane protein function: an energetic perspective. Annu. Rev. Biophys. Biomol. Struct. 36, 107–130 (2007).
Chadda, R. et al. Membrane transporter dimerization driven by differential lipid solvation energetics of dissociated and associated states. eLife 10, e63288 (2021).
Gupta, K. et al. The role of interfacial lipids in stabilizing membrane protein oligomers. Nature 541, 421–424 (2017).
Katta, V. & Chait, B. T. Observation of the heme-globin complex in native myoglobin by electrospray-ionization mass spectrometry. J. Am. Chem. Soc. 113, 8534–8535 (1991).
McKay, A. R., Ruotolo, B. T., Ilag, L. L. & Robinson, C. V. Mass measurements of increased accuracy resolve heterogeneous populations of intact ribosomes. J. Am. Chem. Soc. 128, 11433–11442 (2006).
Heck, A. J. R. Native mass spectrometry: a bridge between interactomics and structural biology. Nat. Methods 5, 927–933 (2008).
Sahasrabuddhe, A. et al. Confirmation of intersubunit connectivity and topology of designed protein complexes by native MS. Proc. Natl Acad. Sci. USA 115, 1268–1273 (2018).
Li, H., Nguyen, H. H., Ogorzalek Loo, R. R., Campuzano, I. D. G. & Loo, J. A. An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes. Nat. Chem. 10, 139–148 (2018).
Skinner, O. S. et al. Top-down characterization of endogenous protein complexes with native proteomics. Nat. Chem. Biol. 14, 36–41 (2018).
Whitelegge, J. P., Gundersen, C. B. & Faull, K. F. Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins. Protein Sci. 7, 1423–1430 (1998).
Barrera, N. P., Di Bartolo, N., Booth, P. J. & Robinson, C. V. Micelles protect membrane complexes from solution to vacuum. Science 321, 243–246 (2008).
Laganowsky, A. et al. Membrane proteins bind lipids selectively to modulate their structure and function. Nature 510, 172–175 (2014).
Cong, X., Liu, Y., Liu, W., Liang, X. & Laganowsky, A. Allosteric modulation of protein-protein interactions by individual lipid binding events. Nat. Commun. 8, 2203 (2017).
Zhang, G., Keener, J. E. & Marty, M. T. Measuring remodeling of the lipid environment surrounding membrane proteins with lipid exchange and native mass spectrometry. Anal. Chem. 92, 5666–5669 (2020).
Fantin, S. M. et al. Ion mobility-mass spectrometry reveals the role of peripheral myelin protein dimers in peripheral neuropathy. Proc. Natl Acad. Sci. USA 118, e2015331118 (2021).
Hopper, J. T. S. et al. Detergent-free mass spectrometry of membrane protein complexes. Nat. Methods 10, 1206–1208 (2013).
Konijnenberg, A. et al. Top-down mass spectrometry of intact membrane protein complexes reveals oligomeric state and sequence information in a single experiment. Protein Sci. 24, 1292–1300 (2015).
Chorev, D. S. et al. Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry. Science 362, 829–834 (2018).
Chen, S. et al. Capturing a rhodopsin receptor signalling cascade across a native membrane. Nature 604, 384–390 (2022).
Sarkis, J. & Vié, V. Biomimetic models to investigate membrane biophysics affecting lipid-protein interaction. Front. Bioeng. Biotechnol. 8, 270 (2020).
Keener, J. E. et al. Chemical additives enable native mass spectrometry measurement of membrane protein oligomeric state within intact nanodiscs. J. Am. Chem. Soc. 141, 1054–1061 (2019).
Cheng, H.-T., Megha & London, E. Preparation and properties of asymmetric vesicles that mimic cell membranes: effect upon lipid raft formation and transmembrane helix orientation. J. Biol. Chem. 284, 6079–6092 (2009).
Richmond, D. L. et al. Forming giant vesicles with controlled membrane composition, asymmetry, and contents. Proc. Natl Acad. Sci. USA 108, 9431–9436 (2011).
Duncan, A. L., Song, W. & Sansom, M. S. P. Lipid-dependent regulation of ion channels and G protein-coupled receptors: insights from structures and simulations. Annu. Rev. Pharmacol. Toxicol. 60, 31–50 (2020).
Redka, D. S. et al. Coupling of G proteins to reconstituted monomers and tetramers of the M2 muscarinic receptor. J. Biol. Chem. 289, 24347–24365 (2014).
Hanson, C. L. et al. Phospholipid complexation and association with apolipoprotein C-II: insights from mass spectrometry. Biophys. J. 85, 3802–3812 (2003).
Frick, M., Schwieger, C. & Schmidt, C. Liposomes as carriers of membrane-associated proteins and peptides for mass spectrometric analysis. Angew. Chem. Int. Ed. 60, 11523–11530 (2021).
Mitchell, M. J. et al. Engineering precision nanoparticles for drug delivery. Nat. Rev. Drug Discov. 20, 101–124 (2021).
Ogorzalek Loo, R. R., Lakshmanan, R. & Loo, J. A. What protein charging (and supercharging) reveal about the mechanism of electrospray ionization. J. Am. Soc. Mass. Spectrom. 25, 1675–1693 (2014).
Going, C. C., Xia, Z. & Williams, E. R. New supercharging reagents produce highly charged protein ions in native mass spectrometry. Analyst 140, 7184–7194 (2015).
Fort, K. L. et al. Expanding the structural analysis capabilities on an Orbitrap-based mass spectrometer for large macromolecular complexes. Analyst 143, 100–105 (2017).
Casares, D., Escribá, P. V. & Rosselló, C. A. Membrane lipid composition: effect on membrane and organelle structure, function and compartmentalization and therapeutic avenues. Int. J. Mol. Sci. 20, 2167 (2019).
Vance, J. E. Phospholipid synthesis and transport in mammalian cells. Traffic 16, 1–18 (2015).
van Meer, G., Voelker, D. R. & Feigenson, G. W. Membrane lipids: where they are and how they behave. Nat. Rev. Mol. Cell Biol. 9, 112–124 (2008).
Weber, T. et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772 (1998).
Rothman, J. E., Krishnakumar, S. S., Grushin, K. & Pincet, F. Hypothesis — buttressed rings assemble, clamp, and release SNAREpins for synaptic transmission. FEBS Lett. 591, 3459–3480 (2017).
Takamori, S. et al. Molecular anatomy of a trafficking organelle. Cell 127, 831–846 (2006).
Kitova, E. N., El-Hawiet, A., Schnier, P. D. & Klassen, J. S. Reliable determinations of protein-ligand interactions by direct ESI-MS measurements. Are we there yet? J. Am. Soc. Mass. Spectrom. 23, 431–441 (2012).
Chingin, K. & Barylyuk, K. Charge-state-dependent variation of signal intensity ratio between unbound protein and protein-ligand complex in electrospray ionization mass spectrometry: the role of solvent-accessible surface area. Anal. Chem. 90, 5521–5528 (2018).
Budelier, M. M. et al. Photoaffinity labeling with cholesterol analogues precisely maps a cholesterol-binding site in voltage-dependent anion channel-1. J. Biol. Chem. 292, 9294–9304 (2017).
Hu, Y., Zhu, L. & Ma, C. Structural roles for the juxtamembrane linker region and transmembrane region of synaptobrevin 2 in membrane fusion. Front. Cell Dev. Biol. 8, 609708 (2020).
François-Martin, C., Rothman, J. E. & Pincet, F. Low energy cost for optimal speed and control of membrane fusion. Proc. Natl Acad. Sci. USA 114, 1238–1241 (2017).
Yang, S.-T., Kreutzberger, A. J. B., Lee, J., Kiessling, V. & Tamm, L. K. The role of cholesterol in membrane fusion. Chem. Phys. Lipids 199, 136–143 (2016).
Linetti, A. et al. Cholesterol reduction impairs exocytosis of synaptic vesicles. J. Cell Sci. 123, 595–605 (2010).
Kreutzberger, A. J. B., Kiessling, V. & Tamm, L. K. High cholesterol obviates a prolonged hemifusion intermediate in fast SNARE-mediated membrane fusion. Biophys. J. 109, 319–329 (2015).
Deng, Z., Yuan, B. & Yang, K. Cardiolipin selectively binds to the interface of VsSemiSWEET and regulates its dimerization. J. Phys. Chem. Lett. 12, 1940–1946 (2021).
Akimov, S. A., Polynkin, M. A., Jiménez-Munguía, I., Pavlov, K. V. & Batishchev, O. V. Phosphatidylcholine membrane fusion is pH-dependent. Int. J. Mol. Sci. 19, 1358 (2018).
Mahal, L. K., Sequeira, S. M., Gureasko, J. M. & Söllner, T. H. Calcium-independent stimulation of membrane fusion and SNAREpin formation by synaptotagmin I. J. Cell Biol. 158, 273–282 (2002).
Krishnakumar, S. S. et al. Conformational dynamics of calcium-triggered activation of fusion by synaptotagmin. Biophys. J. 105, 2507–2516 (2013).
Guan, L., Mirza, O., Verner, G., Iwata, S. & Kaback, H. R. Structural determination of wild-type lactose permease. Proc. Natl Acad. Sci. USA 104, 15294–15298 (2007).
Hariharan, P. et al. Structural and functional characterization of protein–lipid interactions of the Salmonella typhimurium melibiose transporter MelB. BMC Biol. 16, 85 (2018).
Yao, X., Fan, X. & Yan, N. Cryo-EM analysis of a membrane protein embedded in the liposome. Proc. Natl Acad. Sci. USA 117, 18497–18503 (2020).
Marty, M. T. et al. Bayesian deconvolution of mass and ion mobility spectra: from binary interactions to polydisperse ensembles. Anal. Chem. 87, 4370–4376 (2015).
Tsugawa, H. et al. A lipidome atlas in MS-DIAL 4. Nat. Biotechnol. 38, 1159–1163 (2020).
Ellena, J. F. et al. Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation. Proc. Natl Acad. Sci. USA 106, 20306–20311 (2009).
Monticelli, L. et al. The MARTINI coarse-grained force field: extension to proteins. J. Chem. Theory Comput. 4, 819–834 (2008).
de Jong, D. H. et al. Improved parameters for the MARTINI coarse-grained protein force field. J. Chem. Theory Comput. 9, 687–697 (2013).
Periole, X., Cavalli, M., Marrink, S.-J. & Ceruso, M. A. Combining an elastic network with a coarse-grained molecular force field: structure, dynamics, and intermolecular recognition. J. Chem. Theory Comput. 5, 2531–2543 (2009).
Wassenaar, T. A., Ingólfsson, H. I., Böckmann, R. A., Tieleman, D. P. & Marrink, S. J. Computational lipidomics with insane: a versatile tool for generating custom membranes for molecular simulations. J. Chem. Theory Comput. 11, 2144–2155 (2015).
Berendsen, H. J. C., van der Spoel, D. & van Drunen, R. GROMACS: a message-passing parallel molecular dynamics implementation. Comput. Phys. Commun. 91, 43–56 (1995).
Van Der Spoel, D. et al. GROMACS: fast, flexible, and free. J. Comput. Chem. 26, 1701–1718 (2005).
Parrinello, M. Polymorphic transitions in single crystals: a new molecular dynamics method. J. Appl. Phys. 52, 7182 (1981).
de Jong, D. H., Baoukina, S., Ingólfsson, H. I. & Marrink, S. J. Martini straight: boosting performance using a shorter cutoff and GPUs. Comput. Phys. Commun. 199, 1–7 (2016).
Pincet, F. et al. FRAP to characterize molecular diffusion and interaction in various membrane environments. PLoS ONE 11, e0158457 (2016).
Marra, J. Controlled deposition of lipid monolayers and bilayers onto mica and direct force measurements between galactolipid bilayers in aqueous solutions. J. Colloid Interface Sci. 107, 446–458 (1985).
Braga, J., Desterro, J. M. P. & Carmo-Fonseca, M. Intracellular macromolecular mobility measured by fluorescence recovery after photobleaching with confocal laser scanning microscopes. Mol. Biol. Cell 15, 4749–4760 (2004).
Wu, J., Shekhar, N., Lele, P. P. & Lele, T. P. FRAP analysis: accounting for bleaching during image capture. PLoS ONE 7, e42854 (2012).
Kang, M., Andreani, M. & Kenworthy, A. K. Validation of normalizations, scaling, and photofading corrections for FRAP data analysis. PLoS ONE 10, e0127966 (2015).
Acknowledgements
We acknowledge the late R. Kaback for several critical insights on making lipid bilayers. This work was partly supported by the National Institutes of Health grants R01GM141192 to K.G., R01DK027044 to J.E.R. and S.S.K., and R01GM122759 and R21NS105863 to L.G. A.J.W. was funded by a Pembroke College (University of Oxford) Rokos Award. A.L.D. was funded by the Biochemistry Department, University of Oxford, and Pembroke College Oxford.
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A.P. and K.G. designed the research and established the method. A.P. performed all experiments on VAMP2, semiSWEET, and MscL. F.G. performed the experiments on AqpZ, MelB, and LacY. A.L.D. and A.J.W. performed molecular dynamics simulations. C.B. performed the negative-stain EM experiments and data analysis. R.M. performed lipidomics experiments and data analysis. P.H. and L.G. provided the purified MelB and LacY. J.N.D.G performed the FRAP experiments under the supervision of F.P. J.C. prepared the VAMP2 construct. A.P. performed the vesicle fusion experiments under the guidance of S.R. S.K. evaluated and provided critical feedback for bulk fusion experiments. J.E.R. provided supervision on all experiments on VAMP2 and SVs and provided general critical insights. K.G. and A.P. wrote the manuscript with contributions from all other authors.
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Extended data
Extended Data Fig. 1 Flow chart of preparation of native-MS ready proteo-liposomes.
Detail flow chart of two alternate approaches that can yield native-MS ready proteo-liposomes with desired lipid composition and membrane biophysical properties. The formed proteo-liposomes can be isolated from the free protein and the empty liposomes through a floatation assay. For better visualization, we typically add 1% RhodPE in our lipid mix. Post-floatation, the quality of the liposomes is assessed via negative stain imaging and DLS. An optional step can be introduced to control the diameter, and in turn the curvature, of the liposomes by passing them through an extruder fitted with filters of desired dimension. The size is further assessed by negative stain or DLS. Between the two protocols, while float-up method is typically longer, for certain protein-detergent scenario, dialysis can be more effective way to reconstitute. Figure graphics were created using Biorender.com.
Extended Data Fig. 2 Negative stain TEM images of different lipid vesicles.
a–e - Negative stain TEM images of different lipid vesicles which mimics of different eukaryotic organellar membranes. f–h - Negative stain TEM images of different curvature synaptic vesicles. The desired curvature was achieved by passing the synaptic vesicles like lipid vesicles through different pore size membrane. All the sample was taken from a pool of vesicles that were subjected to nativeMS (Fig. 1), confirming the vesicles in the samples. All measurements were replicated over three independent measurements.
Extended Data Fig. 3 Detail assignments of the bound lipids.
In the spectra of a, Golgi like lipid membrane, b, mitochondria like membrane, c, ER like lipid membrane, d, PM like lipid membrane, e, and f, bacterial inner plasma membrane like lipid membrane as observed in the Fig. 1. The identity of the lipids could be determined by directly comparing the adduct masses against the theoretical masses of the individual lipids used to make the bilayer (Supplementary Table 2). All measurements were replicated over three independent measurements.
Extended Data Fig. 4 Detailed lipidomics profile of the extracted E.coli membrane lipids.
Detailed lipidomics profile of the extracted E.coli membrane used to reconstitute LacY and AqpZ (Fig. 1). For a, PE, b, PG, c, CL, and d, LPE, the detected individual lipid components were divided as per the total chain length distribution and quantified. The data presented as mean ± s.e. (N = 2).
Extended Data Fig. 5 nMS analysis of IMP (VAMP2) from bilayer with different fluidity.
a, nMS analysis of IMP (VAMP2) from vesicles with increasing amount of cholesterol. All measurements were replicated over three independent measurements, b, The diffusion coefficient (µm2/s) plotted against the mol% of cholesterol in the bilayer. As can be seen from the plot, with increase in mol% of cholesterol in the bilayer the diffusion coefficient decreases. That means the fluidity of the bilayer decreases with increase in cholesterol in the bilayer. Each data point represents the average diffusion coefficient from three independent experiment. The data presented as mean ± s.e. (N = 3).
Extended Data Fig. 6 Isolation spectra of 4+ charge state lipid bound VAMP2 from synaptic vesicles mimicking vesicles. and Lipid bound/Apo-protein ratio.
a, Isolation spectra of 4+ charge state lipid bound VAMP2 from synaptic vesicles mimicking vesicles. b, Lipid bound/Apo-protein ratio calculated from area under the curve of apo-protein peaks and lipid bound protein peaks (and then total lipid bound area taken to get ratio) in nMS spectra of VAMP2 from synaptic vesicles mimicking vesicles.
Extended Data Fig. 7 Spectra of VAMP2 showing no SM/EPC binding.
Spectra of VAMP2 from SV-like lipid vesicles where all the PC were replaced by a, SM and b, EPC (lipid compositions shown in the respective inset tables). The arrows showing the absence of theoretically predicted SM/EPC bound m/z values. In both the cases, under the same MS conditions, no detectable amount of SM or EPC binding was observed. All measurements were replicated over three independent measurements.
Extended Data Fig. 8 Spectra of detergent solubilized VAMP2 incubated with the same mixture of lipids present in SV shows no specificity of lipid binding.
Spectra of detergent solubilized VAMP2 incubated with the same mixture of lipids present in SV-like liposomes. Taking the example of +4 charge state, the data clearly shows that VAMP2 binds to several phospholipids without any specificity to any specific lipids. Further no detectable amount of cholesterol binding was observed. The data was obtained in MS conditions identical to that used for detecting VAMP 2 from SV like liposomes. All measurements were replicated over three independent measurements.
Extended Data Fig. 9 Bulk fusion assay for the VAMP2.
a, % Fusion vs the time course data for the VAPM2 in SV like and EPC-SV vesicles. In both the cases, CDV (cytosolic domain of VAMP2) was used a negative control. In presence of excess CDV, CDV binds and occupies the VAMP2 binding domains of the t-SNAREs. Consequently, upon addition of VAMP2 reconstituted vesicles, these vesicular VAMP2s cannot bind to t-SNARE anymore and no fusion is observed. As seen, the EPC-SV vesicles, devoid of PC, fuses with the PM at a significantly lower rate than the PC containing native-like SV liposomes. b, SDS-Gel picture showing similar band intensity of VAMP2 in SV like and EPC-SV vesicles. c, Vesicle fusion assay performed between t-SNARE in PM like lipid composition vesicles (tSNARE-PM) and VAMP2 in SV like vesicles (VAMP2-PC). Subsequently, to assess the effect of EPC on fusion rate, PC was replaced with EPC in PM like vesicles containing t-SNARE. Similar rate of fusion was observed as observed in t-SNARE in PM like vesicles with PC. This confirms that the reduced fusion rate observed for SV-EPC vesicles (Fig. 2), is not because of replacing PC with EPC in the bilayer, but due to abrogation of VAMP2-PC binding. The data presented as mean ± s.e (N = 5).
Extended Data Fig. 10 NativeMS spectra of semisweet from a. detergent, b. bilayer with PE, PG without CL, c. bilayer with PE, PG, and 2.5% CL.
The individual peaks were plotted in the Origin software and then the area under the curve was obtained by fitting the peak with constant baseline mode. The 6+ monomer charge state and 9+ and 11+ dimer charge states were considered for oligomer % calculation. The peak areas of protein and lipid-bound protein from Origin software were then taken for percentage calculations and the final bar graph of this calculation is shown as an inset in a. The data clearly shows increase in CL% increases the dimeric population in the bilayer. The inset on the left side in each panel shows the mass plot obtained from UniDec analysis.
Supplementary information
Supplementary Information
Supplementary Tables 1 and 2
Source data
Source Data Fig. 1
Mass spectrometry data in .xlsx format
Source Data Fig. 2
Mass spectrometry data in .xlsx format and numerical data of bulk fusion assay in .xlsx format
Source Data Extended Data Fig. 2
Unprocessed TEM images of vesicles in .jpg and.tif format
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Mass spectrometry data in .xlsx format
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Lipidomics data in .xlsx format
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Mass spectrometry data in .xlsx format and FRAP experimental data in .xlsx format
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Mass spectrometry data in .xlsx format and lipid-bound and apoprotein calculation data in .xlsx format
Source Data Extended Data Fig. 7
Mass spectrometry data in .xlsx format
Source Data Extended Data Fig. 8
Mass spectrometry data in .xlsx format
Source Data Extended Data Fig. 9
Numerical data of bulk fusion assay in .xlsx format and unprocessed, uncropped gel image in .tiff format
Source Data Extended Data Fig. 10
Mass spectrometry data in .xlsx format
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Panda, A., Giska, F., Duncan, A.L. et al. Direct determination of oligomeric organization of integral membrane proteins and lipids from intact customizable bilayer. Nat Methods 20, 891–897 (2023). https://doi.org/10.1038/s41592-023-01864-5
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DOI: https://doi.org/10.1038/s41592-023-01864-5
