title: Multicolor fluorescence-based screening toward structural analysis of multiprotein membrane complexes.
abstract: Structures of membrane protein complexes provide a wealth of information on their biological function, the interplay among their subunits, and on ligand binding. Structural genomics of multiprotein membrane complexes seek to deliver structural information ideally of most of these complexes. Models of abundant native membrane protein complexes have been proposed from X-ray crystallography or single-particle cryo-EM. However, most of the remaining membrane protein complexes persist in very low copy numbers per cell and cannot be isolated from their native source without tremendous efforts. Therefore, heterologous expression systems are continually being developed to overproduce membrane protein complexes in various host cells of bacterial or eukaryotic origin. Still, only a small fraction of membrane proteins is suitable for structure determination due to poor expression levels, misfolding and aggregation, complex heterogeneity, imbalanced stoichiometry, and difficulties in solubilization as well as stabilization of the complexes. Powerful tools are therefore necessary to identify the correct expression host and to validate extraction and purification strategies for a given membrane protein complex at the earliest time point. Here, we discuss a fluorescence-based screening approach particularly tailored for the handy and sensitive analysis of the production and purification process for multiprotein membrane complexes. Multicolor fluorescence-detection size-exclusion chromatography provides a powerful readout system and allows quantitative monitoring of the production of critical single subunits of membrane protein complexes. The approach facilitates the tracking of improvements during sample optimization for monodispersity, balanced stoichiometry, and stability of multisubunit membrane protein complexes.
authors.affiliation: Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Frankfurt/Main, Germany.
authors.forename: Simon
authors.initials: S
authors.lastname: Trowitzsch
authors.affiliation: Institute of Biochemistry, Biocenter, Goethe-University Frankfurt, Frankfurt/Main, Germany; Cluster of Excellence-Macromolecular Complexes, Goethe-University Frankfurt, Frankfurt/Main, Germany. Electronic address: tampe@em.uni-frankfurt.de.
authors.forename: Robert
authors.initials: R
authors.lastname: Tampé
mesh_terms: D000818:Animals; D002850:Chromatography, Gel / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods*; D018360:Crystallography, X-Ray; D003902:Detergents; D005434:Flow Cytometry / Q000379:methods; D005453:Fluorescence; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D010843:Pichia / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D014162:Transfection / Q000379:methods; D014170:Transformation, Genetic
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D046912:Multiprotein Complexes
keywords: ABC transporters; Fluorescence-based screening; Heterologous expression systems; Multicolor fluorescence-detection size-exclusion chromatography; Multisubunit membrane protein complexes; Peptide-loading complex; Structural biology; Transporter associated with antigen processing