## 5 Membrane Proteins in SMALPs
The adenosine receptor expressed in _Pichia pastoris_ or HEK  
Figure 3: Top and side views of high resolution cryo-EM structure of trimeric wild-type AcrB (gray, green, pink subunits) and associated lipids (yellow) in SMA2000 nanodiscs (EMD-7074, PDB ID 6 BAJ) [45]. The central cavity is filled with 24 triangular-like core lipids. 293T cells retains ligand binding and stability after extraction with SMA(2:1). Moreover, after storage and freeze-thawing in SMALPs it remains stable seven times longer than in detergent preparations [14]. Solubilization of the melatonin and ghrelin receptors from _Pichia pastoris_ membranes or liposomes with SMA(2:1) or SMA(3:1) yields 13 nm diameter nanodiscs that retain ligand binding and signal transduction activity [47]. The cannabinoid receptor 1 expressed in insect cells retains its native fold once solubilized with SMA(2:1) based on recognition using conformationally specific antibody in surface plasmon resonance experiments [48]. Moreover, this SMALP'd GPCR is stable and active in yeast and mammalian display formats using fluorescence-activated cell sorting (FACS) even after repeated freeze-thaws. It could be used for screening of libraries of proteins to select for those with enhanced ligand affinity, stability, or potential for targeting membrane protein, demonstrating the potential of the approach for screening for novel biologics. Full length ion channels have been solubilized using SMA(3:1) to reveal the intact pores and symmetric tetramers using negative stain EM [49]. The human hKCNH5 and hKCNQ1 \(\alpha\)-subunits of the human neuronal and cardiac voltage-gated potassium (Kv) channels as well as the regulatory transmembrane KCNE1 \(\beta\)-subunit were SMALP'd from mammalian COS-1 cells and showed better behavior than detergent solubilized forms, being more stable and easier to concentrate without aggregation. The channels are oriented randomly on EM grids, displaying orientations with dimensions in the range of 12-15 nm within the discs. Electron paramagnetic resonance (EPR) studies of a spin-labeled KCNE1 protein solubilized in SMA(3:1) nanodiscs show that sidechains in the aqueous phase are more flexible than those inside the bilayer [50]. This report showed that use of SMA can increase the signal to noise and phase memory time in double electron-electron resonance spectra to yield improved distance measurements. The tetraspanin superfamily represents a technically challenging group of membrane proteins to study because of their small size, oligomerization, multiple disulfides, and glycosylation and palmitoylation groups, as well as the lack of many biochemical assays. Fifteen human tetraspanins were expressed in _S. cerevisiae_, and the five highest yield proteins were solubilized by three different SMA copolymers. The yield of TSPAN7 was comparable to conventional detergents, while CO-029, TSPAN12, and TSPAN18 yields were higher in detergents, and CD63 was resistant to any approach [51]. The variable expression levels and organized superstructure of such proteins emphasize the need for further development of tools for extraction and purification of low abundance memteins and efficient dispersal of very large, ordered networks. NMR studies of SMALPs reveal the structures, dynamics, and interactions of contained transmembrane proteins. The Pf1 bacteriophage coat protein forms stable helical structures in magnetically-oriented 30 nm nanodiscs that are formed with SMA(3:1) [52]. They display sharper NMR signals than those which are obtained in bicelles or peptide-based nanodiscs. The NMR signals of cytochrome b5 protein within magnetically aligned \(\sim\)50 nm nanodiscs made of SMA-EA reveal a stable transmembrane helix [34]. The 34 kDa zinc diffusion facilitator CzcD solubilized with XIRAN 25010 or XIRAN 30010 into 10-15 nm nanodiscs maintains a boundary of bound lipid [53]. The SMALPs display resolvable NMR signals for amide or methyl groups, and NMR assignments can be transferred between solid and solution states.