Identifying and measuring transmembrane helix-helix interactions by FRET.
Identifying and measuring transmembrane helix-helix interactions by FRET.
复制标题
通过 FRET 识别和测量跨膜螺旋-螺旋相互作用。
DOI：
10.1007/978-1-62703-023-6_6
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Lazarova,Tzvetana
中科院分区：
文献类型：
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作者：
Thevenin,Damien;Lazarova,Tzvetana
螺旋跨膜结构域（TM）之间的特异性相互作用在膜蛋白的折叠、稳定性和组装中起着重要作用。因此，它是有吸引力的，以确定螺旋螺旋接触，并寻求在分子水平上的这种相互作用的结构决定因素。在这里，我们提供了一个协议，用于检测和测量特定的螺旋-螺旋相互作用的脂质体通过福斯特共振能量转移（FRET），使用对应于TM结构域的一个完整的膜蛋白的肽。我们给出了一个详细的程序和实用的指导方针，如何设计，制备，处理和表征荧光标记的TM肽在大单层脂质囊泡重建。我们还讨论了FRET测量的一些关键方面，以确保正确的分析和解释光谱数据。我们的方法使用色氨酸/芘作为供体-受体FRET对，但它可以很容易地适应其他荧光对和其他膜模拟环境。识别关键螺旋间接触的能力是研究重要的和实验上具有挑战性的螺旋膜蛋白的稳定性、组装和功能的有价值的工具。
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.