Domanov Yegor A, Molotkovsky Julian G, Gorbenko Galyna P
Department of Biological and Medical Physics, V. N. Karazin Kharkiv National University, 4 Svobody Sq., Kharkiv 61077, Ukraine.
Biochim Biophys Acta. 2005 Oct 1;1716(1):49-58. doi: 10.1016/j.bbamem.2005.09.002.
The method of fluorescence resonance energy transfer (FRET) has been employed to monitor cytochrome c interaction with bilayer phospholipid membranes. Liposomes composed of phosphatidylcholine and varying amounts of anionic lipid cardiolipin (CL) were used as model membranes. Trace amount of fluorescent lipid derivative, anthrylvinyl-phosphatidylcholine was incorporated into the membranes to serve energy donor for heme moiety of cytochrome c. Energy transfer efficiency was measured at different lipid and protein concentrations to obtain extensive set of data, which were further analyzed globally in terms of adequate models of protein adsorption and energy transfer on the membrane surface. It has been found that the cytochrome c association with membranes containing 10 mol% CL can be described in terms of equilibrium binding model (yielding dissociation constant Kd = 0.2-0.4 microM and stoichiometry n = 11-13 lipid molecules per protein binding site) combined with FRET model assuming uniform acceptor distribution with the distance of 3.5-3.6 nm between the bilayer midplane and heme moiety of cytochrome c. However, increasing the CL content to 20 or 40 mol% (at low ionic strength) resulted in a different behavior of FRET profiles, inconsistent with the concepts of equilibrium adsorption of cytochrome c at the membrane surface and/or uniform acceptor distribution. To explain this fact, several possibilities are analyzed, including cytochrome c-induced formation of non-bilayer structures and clusters of charged lipids, or changes in the depth of cytochrome c penetration into the bilayer depending on the protein surface density. Additional control experiments have shown that only the latter process can explain the peculiar concentration dependences of FRET at high CL content.
荧光共振能量转移（FRET）方法已被用于监测细胞色素c与双层磷脂膜的相互作用。由磷脂酰胆碱和不同量的阴离子脂质心磷脂（CL）组成的脂质体被用作模型膜。将痕量的荧光脂质衍生物蒽乙烯基磷脂酰胆碱掺入膜中，作为细胞色素c血红素部分的能量供体。在不同的脂质和蛋白质浓度下测量能量转移效率，以获得大量数据集，并根据膜表面蛋白质吸附和能量转移的适当模型对其进行全局进一步分析。已发现，细胞色素c与含有10 mol% CL的膜的结合可以用平衡结合模型（解离常数Kd = 0.2 - 0.4 microM，化学计量比n =每个蛋白质结合位点11 - 13个脂质分子）结合FRET模型来描述，该模型假设受体均匀分布，双层中平面与细胞色素c的血红素部分之间的距离为3.5 - 3.6 nm。然而，将CL含量增加到20或40 mol%（在低离子强度下）会导致FRET谱的不同行为，这与细胞色素c在膜表面的平衡吸附和/或受体均匀分布的概念不一致。为了解释这一事实，分析了几种可能性，包括细胞色素c诱导的非双层结构和带电脂质簇的形成，或取决于蛋白质表面密度的细胞色素c渗透到双层中的深度变化。额外的对照实验表明，只有后一个过程可以解释高CL含量下FRET的特殊浓度依赖性。
