title: Benchmarking membrane protein detergent stability for improving throughput of high-resolution X-ray structures.
abstract: Obtaining well-ordered crystals is a major hurdle to X-ray structure determination of membrane proteins. To facilitate crystal optimization, we investigated the detergent stability of 24 eukaryotic and prokaryotic membrane proteins, predominantly transporters, using a fluorescent-based unfolding assay. We have benchmarked the stability required for crystallization in small micelle detergents, as they are statistically more likely to lead to high-resolution structures. Using this information, we have been able to obtain well-diffracting crystals for a number of sodium and proton-dependent transporters. By including in the analysis seven membrane proteins for which structures are already known, AmtB, GlpG, Mhp1, GlpT, EmrD, NhaA, and LacY, it was further possible to demonstrate an overall trend between protein stability and structural resolution. We suggest that by monitoring membrane protein stability with reference to the benchmarks described here, greater efforts can be placed on constructs and conditions more likely to yield high-resolution structures.
authors.affiliation: Division of Molecular Biosciences, Membrane Protein Crystallography Group, Imperial College, London SW7 2AZ, UK.
authors.forename: Yo
authors.initials: Y
authors.lastname: Sonoda
authors.forename: Simon
authors.initials: S
authors.lastname: Newstead
authors.forename: Nien-Jen
authors.initials: NJ
authors.lastname: Hu
authors.forename: Yilmaz
authors.initials: Y
authors.lastname: Alguel
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Nji
authors.forename: Konstantinos
authors.initials: K
authors.lastname: Beis
authors.forename: Shoko
authors.initials: S
authors.lastname: Yashiro
authors.forename: Chiara
authors.initials: C
authors.lastname: Lee
authors.forename: James
authors.initials: J
authors.lastname: Leung
authors.forename: Alexander D
authors.initials: AD
authors.lastname: Cameron
authors.forename: Bernadette
authors.initials: B
authors.lastname: Byrne
authors.forename: So
authors.initials: S
authors.lastname: Iwata
authors.forename: David
authors.initials: D
authors.lastname: Drew
mesh_terms: D018360:Crystallography, X-Ray / Q000379:methods* / Q000592:standards; D003902:Detergents / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D055550:Protein Stability; D058767:Protein Unfolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D003902:Detergents; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins