Kinetics of Protein Absorption on Lipid Bilayers

了解驱动和组织蛋白质在生物膜上吸附的机制是阐明膜介导的蛋白质功能的基础。众所周知，细胞色素c是一种小的膜相关蛋白，在线粒体的能量产生机制中发挥着关键作用。最近发现，细胞色素c从线粒体内膜释放是细胞凋亡途径的第一步，细胞色素c主要位于线粒体内膜。线粒体膜内含有丰富的阴离子脂类，但蛋白质的确切功能定位仍然不清楚，尽管过去使用间接方法解决了这一问题。我们的工作人员与NIDDK的Allen Minton博士合作，在低离子条件下检测了蛋白质在阴离子支持的双层膜上的吸附。蛋白质似乎没有吸附到双层表面，但它与双层的相互作用导致了双层流动性的显着增加。这一结果有两个重要的意义：1.在低离子强度条件下，细胞色素c可能插入到阴离子脂双分子膜的疏水核心中，导致双分子膜结构的显著扰动(从而增加了其流动性)。2.在力模式下，原子力显微镜可以用来探测和量化双层的基本热力学性质(屈服强度、线张力、弹性模量等)。我们正在通过继续研究细胞色素c在更接近线粒体内膜的双层上的吸附以及在不同离子条件下的双层的吸附来进一步研究这两种影响；通过用拉曼光谱技术补充AFM来确定蛋白质的存在或不存在，并将给出与相互作用相关的构象/结构变化的指示；以及通过进一步发展AFM技术来测量双层的基本热力学参数。

Understanding the mechanisms that drive and organize protein adsorption onto biological membranes is fundamental to clarifying membrane-mediated protein function. It is known that cytochrome c is a small, membrane-associated protein playing a critical role in the energy production machinery in the mitochondria. Recently, it was found that cytochrome c release from the inner mitochondria membrane, where it primarily resides, is the first step in the apoptotic pathway. The inner mitochondria membrane is rich in anionic lipids but the exact functional localization of the protein remains obscure despite the number of past efforts to resolve the question using indirect methodologies. Our staff, in collaboration with Dr. Allen Minton of NIDDK, examined the adsorption of the protein to anionic supported bilayers under low ionic conditions. It appears that the protein does not adsorb to the bilayer surface yet its interaction with the bilayer results in a significant increase of bilayer fluidity. This result has two important implications: 1. Cytochrome c in low ionic strength conditions probably inserts into the hydrophobic core of anionic lipid bilayers resulting in a significant perturbation of the bilayer structure (hence its increased fluidity). 2. The AFM can be used, in the force mode, to probe and quantify fundamental thermodynamic properties of bilayers (yield strength, line tension, elasticity modulus, etc.). We are examining both implications further by continuing the study of cytochrome c adsorption to bilayers that closer resemble the inner mitochondria membrane as well as bilayers under varying ionic conditions; by complementing the AFM with the Raman spectroscopy technique that will ascertain the presence or absence of the protein and will give indications of conformational/structural changes associated with the interaction; and, by further developing the AFM technique for the measurement of fundamental thermodynamic parameters of bilayers in general.