title: Multicolour fluorescence-detection size-exclusion chromatography for structural genomics of membrane multiprotein complexes.
abstract: Many interesting and important membrane proteins are hetero-oligomeric. However, besides naturally abundant examples, the structures of relatively few such complexes are known. Partly, this is due to difficulties in expression, stoichiometric assembly, and in the evaluation of their stability prior to crystallization trials. Here we describe a new approach, which allows rapid assessment of protein complex quality, assembly and stoichiometry, simplifying the search for conditions conducive to long-term stability and crystallization. Multicolour fluorescence size-exclusion chromatography (MC-FSEC) is used to enable tracking of individual subunits through expression, solubilization and purification steps. We show how the method has been applied to the heterodimeric transporter associated with antigen processing (TAP) and demonstrate how it may be extended in order to analyse membrane multisubunit assemblies.
authors.affiliation: Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Frankfurt am Main, Germany.
authors.forename: David
authors.initials: D
authors.lastname: Parcej
authors.forename: Renate
authors.initials: R
authors.lastname: Guntrum
authors.forename: Sabine
authors.initials: S
authors.lastname: Schmidt
authors.forename: Andreas
authors.initials: A
authors.lastname: Hinz
authors.forename: Robert
authors.initials: R
authors.lastname: Tampé
mesh_terms: D002850:Chromatography, Gel; D003116:Color; D003902:Detergents / Q000737:chemistry; D005822:Genetic Vectors / Q000235:genetics; D023281:Genomics / Q000379:methods*; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins