bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles

Abstract

In the past, structural and functional studies of voltagegated ion channels have provided profound insights into their function MacKinnon, 2003;Miller, 2003;Tombola et al., 2006Tombola et al., , 2007Long et al., 2007;Bezanilla, 2008; Villalba-Galea et al., 2009a,b;Catterall and Yarov-Yarovoy, 2010;Tao et al., 2010;DeCaen et al., 2011;Payandeh et al., 2011Payandeh et al., , 2012Vargas et al., 2011;Catterall, 2012;Jiang and Gonen, 2012). Most of the past studies assumed that the lipids around voltage-gated ion channels act as a solvent for their hydrophobic transmembrane domains, whereas *H. Zheng and S. Lee contributed equally to this paper. Correspondence to Qiu-Xing Jiang: q i u -x i n g . j i a n g @ u t s o u t h w e s t e r n . e d u or qxjiang@ufl.edu M.C. Llaguno's present address is Dept. of Cell Biology, Yale University, New Haven, CT 06510.Q.-X. Jiang's present address is Dept. of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611.Abbreviations used in this paper: AB-NTA, N-(5-amino-1-carboxypentyl) iminodiacetic acid; BLM, bilayer lipid membrane; bSUM, bead-supported unilamellar membrane; cryoEM, cryo-electron microscopy; EDC, 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride; Fv, a variable fragment of the 33H1 antibody; GFP, green fluorescent protein; Kv, voltagegated potassium; MTS-PEG5K, methoxy-poly-(ethylene glycol)-5000 amidoproprionyl methanethiosulfonate; Sulfo-NHS, N-hydroxysulfosuccinimide. the proteinaceous parts are solely responsible for voltagedependent function. Recent studies, however, recognize that lipids play critically important roles in the controlled opening or closing (gating) of these ion channels (Ramu et al., 2006;Schmidt et al., 2006;Schmidt and MacKinnon, 2008;Xu et al., 2008;Zheng et al., 2011;Jiang and Gonen, 2012;Brohawn et al., 2014;Hite et al., 2014). Compelling evidence from these studies suggests that lipids surrounding a voltage-gated potassium (Kv) channel exert strong gating effects on the channels by directly affecting the energetics behind the conformational changes of the voltage-sensor domains. We observed that removal of the phosphate groups in the lipid bilayers around a Kv channel directly switches its voltage-sensor domains into the "DOWN" (also called the "resting" or "hyperpolarized") conformation in the absence of any change in transmembrane electrostatic potential. We named this phenomenon the "lipid-dependent bSUM: A bead-supported unilamellar membrane system facilitating unidirectional insertion of membrane proteins into giant vesicles Fused or giant vesicles, planar lipid bilayers, a droplet membrane system, and planar-supported membranes have been developed to incorporate membrane proteins for the electrical and biophysical analysis of such proteins or the bilayer properties. However, it remains difficult to incorporate membrane proteins, including ion channels, into reconstituted membrane systems that allow easy control of operational dimensions, incorporation orientation of the membrane proteins, and lipid com...

Current problems and future avenues in proteoliposome research

“…Other approaches for guided orientation employed Ni-NTA-functionalized beads to immobilize His-tagged MPs prior to reconstitution, [96][97][98] a method also used to form planar bilayers for AFM studies. 99,100 The liposomal membrane was formed de novo between the immobilized MPs either around the bead-support, 96 or the beads were suggested to force unidirectional orientation because they were too big to be incorporated into the newly formed liposomes. 97,98 However, a more thorough characterization of these methods, e.g.…”

“…Approaches, in which orientation is guided by steric constraints might display a promising tool that has to be further developed in the future. [95][96][97][98] Robust and easy to implement methods for the quantitative determination of incorporation yield and relative protein orientation are required to compare established and develop new reconstitution protocols, e.g. only a small subset of detergents have been used in reconstitution.…”

“…4 B) The calculated distribution of proteoliposome populations after reconstitution with a 1:1 liposome to protein stoichiometry and a 70% green-side out preference in orientation. insertion, 85,94 by coupling the MP to a solid support (a) that will be encapsulated by the liposome 96 or by attaching a fusion domain 95 or coupling to a solid support (b) 97,98 that will be excluded from the vesicle interior. A) MPs embedded in a polymerosome membrane enables import and export of substrates and products from a synthetic reaction cell.…”

Application of polymersomes in membrane protein study and drug discovery: Progress, strategies, and perspectives

“…To induce immune-modulatory response, it is essential for MHC-I proteins to be expressed on the extracellular-side of antigen-presenting self-assembly of block copolymers (Figure 6d). 111 After confirming the The presence of natural peptide anchors allows the tethering of water-soluble protein or enzyme to membranes. These natural peptides are reconstituted into PMOXA-PDMS-PMOXA polymersomes.…”

“…94,114 Polymersomes without MPs, as well as excess cell-free expression reaction reagents, can be removed from proteopolymersomes by methods similar to detergent removal including dialysis, 110 gel filtration or SEC, 86,87 centrifugation, 53 and bio-beads mediated process. 111 A limitation of the direct incorporate approach is that the necessary posttranslational modifications, which are required for the formation of fully functional proteins may not occur, unless known enzymes responsible for these processes are added to the reaction mixture. 123…”