Thévenin Damien, Lazarova Tzvetana
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA.
Methods Mol Biol. 2012;914:87-106. doi: 10.1007/978-1-62703-023-6_6.
Specific interactions between helical transmembrane domains (TMs) play essential roles in the mechanisms governing the folding, stability and assembly of integral membrane proteins. Thus, it is appealing to identify helix-helix contacts and to seek the structural determinants of such interactions at the molecular level. Here, we provide a protocol for detecting and measuring specific helix-helix interactions in liposomes by Förster resonance energy transfer (FRET), using peptides corresponding to the TM domains of an integral membrane protein. We give a detailed procedure and practical guidelines on how to design, prepare, handle, and characterize fluorescently labeled TM peptides reconstituted in large unilamellar lipid vesicles. We also discuss some critical aspects of FRET measurements to ensure the correct analysis and interpretation of spectral data. Our method uses tryptophan/pyrene as the donor-acceptor FRET pair, but it can be easily adapted to other fluorescence pairs and to other membrane mimetic environments. The ability to identify crucial interhelical contacts is a valuable tool for the study of the stability, assembly, and function of the important and experimentally challenging helical membrane proteins.
螺旋跨膜结构域（TMs）之间的特异性相互作用在决定整合膜蛋白的折叠、稳定性及组装机制中起着至关重要的作用。因此，识别螺旋-螺旋接触并在分子水平探寻此类相互作用的结构决定因素很有吸引力。在此，我们提供了一种利用与整合膜蛋白TM结构域对应的肽段，通过Förster共振能量转移（FRET）检测和测量脂质体中特异性螺旋-螺旋相互作用的方法。我们给出了关于如何设计、制备、处理及表征重构于大单层脂质囊泡中的荧光标记TM肽段的详细步骤和实用指南。我们还讨论了FRET测量的一些关键方面，以确保对光谱数据进行正确的分析和解读。我们的方法使用色氨酸/芘作为供体-受体FRET对，但它可轻松适配于其他荧光对及其他膜模拟环境。识别关键螺旋间接触的能力是研究重要且实验上具有挑战性的螺旋膜蛋白的稳定性、组装及功能的宝贵工具。
