Khadria Ambalika, Senes Alessandro
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, USA.
Methods Mol Biol. 2013;1063:19-36. doi: 10.1007/978-1-62703-583-5_2.
Present day understanding of the thermodynamic properties of integral membrane proteins (IMPs) lags behind that of water-soluble proteins due to difficulties in mimicking the physiological environment of the IMPs in order to obtain a reversible folded system. Despite such challenges faced in studying these systems, significant progress has been made in the study of the oligomerization of single span transmembrane helices. One of the primary methods available to characterize these systems is based on Förster resonance energy transfer (FRET). FRET is a widely used spectroscopic tool that provides proximity data that can be fitted to obtain the energetics of a system. Here we discuss various technical aspects related to the application of FRET to study transmembrane peptide oligomerization in liposomes. The analysis is based on FRET efficiency relative to the concentration of the peptides in the bilayer (peptide:lipid ratio). Some important parameters that will be discussed include labeling efficiency, sample homogeneity, and equilibration. Furthermore, data analysis has to be performed keeping in mind random colocalization of donors and acceptors in liposome vesicles.
由于难以模拟整合膜蛋白（IMPs）的生理环境以获得可逆折叠系统，目前对整合膜蛋白热力学性质的理解落后于水溶性蛋白。尽管在研究这些系统时面临诸多挑战，但在单跨膜螺旋寡聚化的研究方面已取得显著进展。表征这些系统的主要方法之一是基于Förster共振能量转移（FRET）。FRET是一种广泛使用的光谱工具，可提供能通过拟合获得系统能量学的邻近数据。在此，我们讨论与应用FRET研究脂质体中跨膜肽寡聚化相关的各种技术方面。分析基于相对于双层中肽浓度（肽：脂质比）的FRET效率。将讨论的一些重要参数包括标记效率、样品均一性和平衡。此外，在进行数据分析时必须考虑脂质体囊泡中供体和受体的随机共定位。
