Marino Jacopo, Bordag Natalie, Keller Sandro, Zerbe Oliver
Department of Chemistry, University of Zürich, Switzerland.
Protein Sci. 2015 Jan;24(1):38-48. doi: 10.1002/pro.2582. Epub 2014 Oct 25.
The interaction of the Bacillus subtilis protein Mistic with the bacterial membrane and its role in promoting the overexpression of other membrane proteins are still matters of debate. In this study, we aimed to determine whether individual helical fragments of Mistic are sufficient for its interaction with membranes in vivo and in vitro. To this end, fragments encompassing each of Mistic's helical segments and combinations of them were produced as GFP-fusions, and their cellular localization was studied in Escherichia coli. Furthermore, peptides corresponding to the four helical fragments were synthesized by solid-phase peptide synthesis, and their ability to acquire secondary structure in a variety of lipids and detergents was studied by circular dichroism spectroscopy. Both types of experiments demonstrate that the third helical fragment of Mistic interacts only with LDAO micelles but does not partition into lipid bilayers. Interestingly, the other three helices interact with membranes in vivo and in vitro. Nevertheless, all of these short sequences can replace full-length Mistic as N-terminal fusions to achieve overexpression of a human G-protein-coupled receptor in E. coli, although with different effects on quantity and quality of the protein produced. A bioinformatic analysis of the Mistic family expanded the number of homologs from 4 to 20, including proteins outside the genus Bacillus. This information allowed us to discover a highly conserved Shine-Dalgarno sequence in the operon mstX-yugO that is important for downstream translation of the potassium ion channel yugO.
枯草芽孢杆菌蛋白Mistic与细菌膜的相互作用及其在促进其他膜蛋白过表达中的作用仍是一个有争议的问题。在本研究中，我们旨在确定Mistic的单个螺旋片段在体内和体外是否足以与膜相互作用。为此，制备了包含Mistic每个螺旋片段及其组合的片段作为GFP融合蛋白，并在大肠杆菌中研究了它们的细胞定位。此外，通过固相肽合成法合成了对应于四个螺旋片段的肽，并通过圆二色光谱研究了它们在各种脂质和去污剂中获得二级结构的能力。这两种类型的实验均表明，Mistic的第三个螺旋片段仅与LDAO胶束相互作用，但不分配到脂质双层中。有趣的是，其他三个螺旋在体内和体外均与膜相互作用。然而，所有这些短序列都可以作为N端融合蛋白替代全长Mistic，以在大肠杆菌中实现人G蛋白偶联受体的过表达，尽管对产生的蛋白质的数量和质量有不同影响。对Mistic家族的生物信息学分析将同源物的数量从4个扩展到20个，包括芽孢杆菌属以外的蛋白质。这些信息使我们能够在操纵子mstX-yugO中发现一个高度保守的Shine-Dalgarno序列，该序列对钾离子通道yugO的下游翻译很重要。
