title: Production of disulfide-stabilized transmembrane peptide complexes for structural studies.
abstract: Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.
authors.affiliation: Structural Biology Division, The Walter and Eliza Hall Institute of Medical Research, Australia.
authors.forename: Pooja
authors.initials: P
authors.lastname: Sharma
authors.forename: Mariam
authors.initials: M
authors.lastname: Kaywan-Lutfi
authors.forename: Logesvaran
authors.initials: L
authors.lastname: Krshnan
authors.forename: Eamon F X
authors.initials: EF
authors.lastname: Byrne
authors.forename: Melissa Joy
authors.initials: MJ
authors.lastname: Call
authors.forename: Matthew Edwin
authors.initials: ME
authors.lastname: Call
mesh_terms: D004220:Disulfides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004220:Disulfides; D008565:Membrane Proteins; D011994:Recombinant Proteins