Páli Tibor, Kóta Zoltán
Institute of Biophysics, Biological Research Centre, Szeged, Hungary.
Methods Mol Biol. 2013;974:297-328. doi: 10.1007/978-1-62703-275-9_14.
Spin label electron paramagnetic resonance (EPR) of lipid-protein interactions reveals crucial features of the structure and assembly of integral membrane proteins. Spin label EPR spectroscopy is the technique of choice to characterize the protein-solvating lipid shell in its highly dynamic nature, because the EPR spectra of lipids that are spin labeled close to the terminal methyl end of their acyl chains display two spectral components, those corresponding to lipids directly contacting the protein and those corresponding to lipids in the bulk fluid bilayer regions of the membrane. In this chapter, typical spin label EPR procedures are presented that allow determination of the stoichiometry of interaction of spin-labeled lipids with the intra-membranous region of membrane proteins or polypeptides, as well as the association constant of the spin-labeled lipid with respect to the host lipid. The lipids giving rise to the so-called immobile spectral component in the EPR spectrum of such samples are identified as the motionally restricted first-shell lipids solvating membrane proteins in biomembranes. Stoichiometry and selectivity are directly related to the structure of the intra-membranous sections of membrane-associated proteins or polypeptides and can be used to study the state of assembly of such proteins in the membrane. Since these characteristics of lipid-protein interactions are discussed in detail in the literature [see Marsh (Eur Biophys J 39:513-525, 2010) for a most recent review], here we focus more on how to spin label model and biomembranes and how to measure and analyze the two-component EPR spectra of spin-labeled lipids in phospholipid bilayers that contain proteins or polypeptides. After a description of how to prepare spin-labeled model and native biological membranes, we present the reader with computational procedures for determining the molar fraction of motionally restricted lipids when both, one, or none of the pure isolated-mobile or immobile-spectral components are available. With these topics, this chapter complements a recent methodological paper [Marsh (Methods 46:83-96, 2008)]. The interpretation of the data is discussed briefly, as well as other relevant and recent spin label EPR techniques for studying lipid-protein interactions, not only from the point of view of lipid chain dynamics.
脂质 - 蛋白质相互作用的自旋标记电子顺磁共振（EPR）揭示了整合膜蛋白结构和组装的关键特征。自旋标记EPR光谱法是表征具有高度动态性质的蛋白质溶剂化脂质壳的首选技术，因为在其酰基链末端甲基附近进行自旋标记的脂质的EPR光谱显示出两个光谱成分，一个对应于直接与蛋白质接触的脂质，另一个对应于膜的本体流体双层区域中的脂质。在本章中，将介绍典型的自旋标记EPR程序，这些程序可用于确定自旋标记脂质与膜蛋白或多肽的膜内区域的相互作用化学计量，以及自旋标记脂质相对于主体脂质的缔合常数。在此类样品的EPR光谱中产生所谓固定光谱成分的脂质被鉴定为生物膜中使膜蛋白溶剂化的运动受限的第一壳层脂质。化学计量和选择性与膜相关蛋白或多肽的膜内部分的结构直接相关，可用于研究此类蛋白在膜中的组装状态。由于脂质 - 蛋白质相互作用的这些特征在文献中已有详细讨论[见Marsh（《欧洲生物物理杂志》39:513 - 525，2010年）的最新综述]，因此在这里我们更关注如何对模型膜和生物膜进行自旋标记，以及如何测量和分析含有蛋白质或多肽的磷脂双层中自旋标记脂质的双组分EPR光谱。在描述了如何制备自旋标记的模型膜和天然生物膜之后，我们向读者介绍了在纯分离的移动或固定光谱成分都不存在、只有一个存在或两个都存在时，用于确定运动受限脂质摩尔分数的计算程序。通过这些主题，本章补充了最近的一篇方法学论文[Marsh（《方法》46:83 - 96，2008年）]。还简要讨论了数据的解释，以及其他用于研究脂质 - 蛋白质相互作用的相关和最新的自旋标记EPR技术，不仅从脂质链动力学的角度进行讨论。
