The importance of the membrane for biophysical measurements

Abstract

Within cell membranes numerous protein assemblies reside. Among their many functions, these assemblies regulate the movement of molecules between membranes, facilitate signaling into and out of cells, allow movement of cells by cell-matrix attachment, and regulate the electric potential of the membrane. With such critical roles, membrane protein complexes are of considerable interest for human health, yet they pose an enduring challenge for structural biologists because it is difficult to study these protein structures at atomic resolution in in situ environments. To advance structural and functional insights for these protein assemblies, membrane mimetics are typically employed to recapitulate some of the physical and chemical properties of the lipid bilayer membrane. However, extraction from native membranes can sometimes change the structure and lipid-binding properties of these complexes, leading to conflicting results and fueling a drive to study complexes directly from native membranes. Here we consider the co-development of membrane mimetics with technological breakthroughs in both cryo-electron microscopy (cryo-EM) and native mass spectrometry (nMS). Together, these developments are leading to a plethora of high-resolution protein structures, as well as new knowledge of their lipid interactions, from different membrane-like environments.

Fig. 1. The resolution revolution has transformed EM and MS.

a Cryo-EM structures of bovine F-type ATP synthase from 2012 (modelled from EMD-2091), 2015 (modelled from EMD-3164) and 2019 (reproduced with permission from ), from left to right respectively. Mass spectrum of the V/A-type atpase from Thermus thermophilus recorded in 2011 in DDM micelles with a mass consistent with lipid binding in the centre of the V_o membrane ring. Inset (purple box) mass spectrum of bovine F-type ATP synthase ejected from native membranes in 2018 with mass consistent with the presence of subunits e and 6.8 PL (orange) shown schematically in the structure of porcine ATP synthase b Mass spectrum of complex IV (Cco) recorded in 2015 in C₈E₄ detergent micelles reveals an equilibrium between monomeric (blue) and dimeric forms (grey). The broadness of the peaks is attributed to multiple lipids binding to the complex, forming a lipid plug between the two monomers. By contrast monomeric and dimeric forms ejected directly from membrane vesicles in 2018 yield well-resolved charge states in the case of the monomeric form (blue box, full width at half maximum = 3.8 m/z), with peak broadening for the dimer (grey box) (full width at half maximum=32.8 m/z). The structure is modelled from PDB:2OCC. % is relative intensity, CV is complex V. (CIV)₁ is complex IV monomer, (CIV)₂ is complex IV dimer. Each collored peak series represents an annotated protein complex.

Fig. 2. From micelles to membranes.

The transition from detergent micelles through the various mimetics that have been developed over the last few decades, each with the overall goal of extracting the protein and reconstituting as closely as possible to its original membrane environment. The plethora of membrane protein structures coming to the fore can also be attributed to the new ways of stabilizing these protein complexes in membrane mimetics introduced in recent years. There now exists a wide range of such mimetics including nanodiscs, synthetic polymers, amphipols, peptidiscs and saposins complimenting more established approaches of bicelles, liposomes and vesicles. Each has their merits and compatibilities with various methods but rarely are they universal. For example bicelles, to which membrane protein extracted in detergents, that is followed by encapsulation in lipids flanked by detergent to mimic the membrane, are compatible with NMR. Nanodiscs are often selected for structure determination via EM and peptidiscs are employed with affinity preparations. Moreover, while all are capable of capturing aspects of the membrane environment, inherent differences in their properties lead to more ready application for certain types of protein complexes or applications.

Fig. 3. The effect of detergents on lipid binding and structural integrity.

(a) Comparison of mass spectra of Complex I (CI) in detergent micelles (upper) and directly from vesicles (lower). Upper panel is mass spectrum recorded in a n-Dodecyl-β-D-Maltopyranoside (DDM) detergent micelle, which reveals two series of peaks (labeled orange and purple) differing in mass by 26,612 Da assigned to NDUFS3 (in green, within the protein model). The structure shown is from PDB:5LDW. Both series are assigned to the complete membrane region. The charge states differ between both conditions- the top panel is measured in 2x critical micellar concentration (CMC) of DDM, the bottom panel is CI lacking the N module ejected directly from native membranes with no recourse to detergents or other membrane mimetics. The lipid binding properties change in terms of both the extent and identity of bound lipids. However, the measured mass remains essentially the same for both the detergent extracted and SoLVe measurements (782,772 ± 35 Da compared to 782,478 ± 27 Da) for the membrane arm without NDUFS3 (orange series) and 809,269 ± 47 compared to 809,101 ± 57 Da for the membrane arm with NDUFS3 (purple series) respectively. (b) Comparison of UraA:Proton symporter in β-NG, PDB: 3QE7 (i) whereby the dimer interface is deformed by the protrusion of the acyl chain of the detergent molecules between key helices in each monomer, and in fos-choline 9 and 11 from PDB:5XLS (ii) showing a functional dimer.

Fig. 4. Variation in the subunit stoichiometry of the the Patched receptor, Bam and Ton complexes.

For the patched receptor (green panel) three different EM structures have been reported. First a monomeric structure from DDM micelles in August 2018 (i) followed by a dimeric structure from amphipols in November 2018 (reproduced with permission from) (ii) and a tetrameric structure in May 2019 from GDN micelles (iii). The Bam complex (blue panel) was first reported in 2016, using x-ray crystallography and C₈E₄ micelles shown to contain 4 subunits - BamACDE (i) later reported as a pentamer (BamABCDE) by means of cryo-EM (ii) and using native MS a hexamer of Bam containing two E subunits could also be assigned, both from native membranes and recombinant sources (iii). The Ton complex (orange panel) was shown from x-ray structures to exist in two forms 6ExbB: 3ExbD and 5ExbB:1ExbD (i-ii) and a stoichiometry of 5ExbB:2ExbD was deduced from EM (iii). When ejected from native membranes into a mass spectrometer it was assigned as 5ExbB:1ExbD (ii).