Polar Interactions Trump Hydrophobicity in Stabilizing the Self-Inserting Membrane Protein Mistic
Polar Interactions Trump Hydrophobicity in Stabilizing the Self-Inserting Membrane Protein Mistic
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DOI：
10.1021/ja5064795
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发表时间：
2014-10-01
影响因子：
15
通讯作者：
Keller, Sandro
中科院分区：
文献类型：
--
作者：
Broecker, Jana;Fiedler, Sebastian;Keller, Sandro
典型的整合膜蛋白通过疏水性跨膜螺旋附着在脂质双层上，其拓扑发生需要复杂的插入机制。相比之下，由于进化或功能原因，膜蛋白不能依赖这些机制，需要求助于疏水性以外的驱动力。一个引人注目的例子是自插入枯草芽孢杆菌蛋白 Mistic，它参与生物膜形成，并已被用作支持重组生产和其他膜蛋白双层插入的融合标签。尽管这种不寻常的蛋白质含有大量极性和带电残基，并且缺乏特征性的膜相互作用基序，但它在体内与膜紧密结合，在体外与膜模拟系统紧密结合。因此，我们着手量化极性和非极性相互作用对 Mistic 耦合折叠和插入的贡献。为此，我们定义了蛋白质可以在两种状态平衡下从各种去污剂胶束中完全且可逆地解折叠的条件，并且解折叠状态独立于用于溶解折叠状态的去污剂。这使得以前用于可溶性和β-桶膜蛋白的平衡展开实验成为可能，揭示了与离子和两性离子头基的极性相互作用以及界面偶极电位比与胶束核心的非极性相互作用更有效地稳定蛋白质。这些发现揭示了允许蛋白质与膜模拟环境紧密相互作用而没有主要疏水贡献的力，并合理化了去垢剂对 Mistic 标记膜蛋白的提取和溶解的不同适用性。
Canonical integral membrane proteins are attached to lipid bilayers through hydrophobic transmembrane helices, whose topogenesis requires sophisticated insertion machineries. By contrast, membrane proteins that, for evolutionary or functional reasons, cannot rely on these machineries need to resort to driving forces other than hydrophobicity. A striking example is the self-inserting Bacillus subtilis protein Mistic, which is involved in biofilm formation and has found application as a fusion tag supporting the recombinant production and bilayer insertion of other membrane proteins. Although this unusual protein contains numerous polar and charged residues and lacks characteristic membrane interaction motifs, it is tightly bound to membranes in vivo and membrane-mimetic systems in vitro. Therefore, we set out to quantify the contributions from polar and nonpolar interactions to the coupled folding and insertion of Mistic. To this end, we defined conditions under which the protein can be unfolded completely and reversibly from various detergent micelles by urea in a two-state equilibrium and where the unfolded state is independent of the detergent used for solubilizing the folded state. This enabled equilibrium unfolding experiments previously used for soluble and beta-barrel membrane proteins, revealing that polar interactions with ionic and zwitterionic headgroups and, presumably, the interfacial dipole potential stabilize the protein much more efficiently than nonpolar interactions with the micelle core. These findings unveil the forces that allow a protein to tightly interact with a membrane-mimetic environment without major hydrophobic contributions and rationalize the differential suitability of detergents for the extraction and solubilization of Mistic-tagged membrane proteins.