title: Reconstitution of proteins on electroformed giant unilamellar vesicles.
abstract: In vitro reconstitution of simplified biological systems from molecular parts has proven to be a powerful method for investigating the biochemical and biophysical principles underlying cellular processes. In recent years, there has been a growing interest in reconstitution of protein-membrane interactions to understand the critical role played by membranes in organizing molecular-scale events into micron-scale patterns and protrusions. However, while all reconstitution experiments depend on identifying and isolating an essential set of soluble biomolecules, such as proteins, DNA, and RNA, reconstitution of membrane-based processes involves the additional challenge of forming and working with lipid bilayer membranes with composition, fluidity, and mechanical properties appropriate for the process at hand. Here we discuss a selection of methods for forming synthetic lipid bilayer membranes and present a versatile electroformation protocol that our lab uses for reconstituting proteins on giant unilamellar vesicles. This synthetic membrane-based approach to reconstitution offers the ability to study protein organization and activity at membranes under more cell-like conditions, addressing a central challenge to accomplishing the grand goal of "building the cell."
authors.affiliation: Department of Bioengineering & Biophysics Program, University of California, Berkeley, CA, USA.
authors.forename: Eva M
authors.initials: EM
authors.lastname: Schmid
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany.
authors.forename: David L
authors.initials: DL
authors.lastname: Richmond
authors.affiliation: Department of Bioengineering & Biophysics Program, University of California, Berkeley, CA, USA; Physical Biosciences, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Fletcher
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D008051:Lipid Bilayers / Q000138:chemical synthesis* / Q000378:metabolism*; D018613:Microscopy, Confocal; D011485:Protein Binding / Q000502:physiology; D011506:Proteins / Q000378:metabolism; D053835:Unilamellar Liposomes / Q000138:chemical synthesis* / Q000378:metabolism*
chemical_list: D008051:Lipid Bilayers; D011506:Proteins; D053835:Unilamellar Liposomes; D004247:DNA
keywords: Electroformation; Lipid bilayer; Liposome; Membrane protein; Membrane reconstitution; Protein–membrane interaction; Vesicle; Video protocol