title: Development of a Thermofluor assay for stability determination of membrane proteins using the Na(+)/H(+) antiporter NhaA and cytochrome c oxidase.
abstract: Crystallization of membrane proteins is very laborious and time-consuming, yielding well diffracting crystals in only a minority of projects. Therefore, a rapid and easy method is required to optimize the conditions for initial crystallization trials. The Thermofluor assay has been developed as such a tool. However, its applicability to membrane proteins is still limited because either large hydrophilic extramembranous regions or cysteine residues are required for the available dyes to bind and therefore act as reporters in this assay. No probe has been characterized to discriminate between the hydrophobic surfaces of detergent micelles, folded and detergent-covered membrane proteins and denatured membrane proteins. Of the four dyes tested, the two dyes 1-anilinonaphthalene-8-sulfonic acid (ANS) and SYPRO Orange were systematically screened for compatibility with five detergents commonly used in the crystallization of membrane proteins. ANS showed the weakest interactions with all of the detergents screened. It was possible to determine the melting temperature of the sodium ion/proton antiporter NhaA, a small membrane protein without large hydrophilic domains, over a broad pH range using ANS. Furthermore, cytochrome c oxidase (CcO) was used to apply the method to a four-subunit membrane protein complex. It was possible to obtain preliminary information on the temperature-dependent denaturation of this complex using the dye ANS. Application of the dye 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM) to CcO in the Thermofluor assay enabled the determination of the melting temperatures of distinct subunits of the complex.
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt, Germany.
authors.forename: Martin
authors.initials: M
authors.lastname: Kohlstaedt
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt, Germany.
authors.forename: Iris
authors.initials: I
authors.lastname: von der Hocht
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Hilbers
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Thielmann
authors.affiliation: Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt, Germany.
authors.forename: Hartmut
authors.initials: H
authors.lastname: Michel
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry*; D018360:Crystallography, X-Ray; D003902:Detergents / Q000737:chemistry*; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D010231:Paracoccus denitrificans / Q000201:enzymology; D011487:Protein Conformation; D011489:Protein Denaturation; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry* / Q000378:metabolism; D013696:Temperature
chemical_list: D000817:Anilino Naphthalenesulfonates; D003902:Detergents; D005456:Fluorescent Dyes; D017923:Sodium-Hydrogen Exchangers; C027132:1-anilino-8-naphthalenesulfonate; D003576:Electron Transport Complex IV
keywords: Thermofluor assay; differential scanning calorimetry; membrane proteins