Kohlstaedt Martin, von der Hocht Iris, Hilbers Florian, Thielmann Yvonne, Michel Hartmut
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, 60438 Frankfurt, Germany.
Acta Crystallogr D Biol Crystallogr. 2015 May;71(Pt 5):1112-22. doi: 10.1107/S1399004715004058. Epub 2015 Apr 24.
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.
膜蛋白结晶非常费力且耗时，只有少数项目能得到衍射良好的晶体。因此，需要一种快速简便的方法来优化初始结晶试验的条件。热荧光分析已作为这样一种工具被开发出来。然而，它对膜蛋白的适用性仍然有限，因为现有的染料需要大的亲水性膜外区域或半胱氨酸残基才能结合，从而在该分析中作为报告分子。目前还没有一种探针能够区分去污剂胶束、折叠且被去污剂覆盖的膜蛋白以及变性膜蛋白的疏水表面。在测试的四种染料中，对两种染料1-苯胺基萘-8-磺酸（ANS）和SYPRO Orange进行了系统筛选，以确定它们与膜蛋白结晶中常用的五种去污剂的兼容性。ANS与所有筛选的去污剂的相互作用最弱。使用ANS可以在很宽的pH范围内测定钠离子/质子反向转运蛋白NhaA（一种没有大亲水性结构域的小膜蛋白）的解链温度。此外，细胞色素c氧化酶（CcO）被用于将该方法应用于一种四亚基膜蛋白复合物。使用染料ANS可以获得关于该复合物温度依赖性变性的初步信息。在热荧光分析中，将染料7-二乙氨基-3-（4'-马来酰亚胺基苯基）-4-甲基香豆素（CPM）应用于CcO，能够测定该复合物不同亚基的解链温度。
