Kinetics of Protein Absorption on Lipid Bilayers

了解驱动和组织蛋白质吸附到生物膜上的机制是阐明膜介导的蛋白质功能的基础。已知细胞色素c是一种小的膜相关蛋白，在线粒体的能量产生机制中起关键作用。最近，人们发现，细胞色素c从线粒体内膜释放，它主要驻留，是在凋亡途径的第一步。内线粒体膜富含阴离子脂质，但蛋白质的确切功能定位仍然模糊，尽管过去的努力，以解决这个问题，使用间接的方法。我们的工作人员与NIDDK的艾伦明顿博士合作，研究了在低离子条件下蛋白质对阴离子支撑双层的吸附。似乎蛋白质不吸附到双层表面，但其与双层的相互作用导致双层流动性的显著增加。这一结果有两个重要的意义：1.细胞色素c在低离子强度条件下可能插入到阴离子脂质双层的疏水核心，导致双层结构的显著扰动（因此其流动性增加）。2.原子力显微镜可用于，在力模式下，探测和量化的基本热力学性质的双层（屈服强度，线张力，弹性模量等）。我们正在进一步研究这两种影响，通过继续研究细胞色素c吸附到更接近线粒体内膜的双层以及在不同离子条件下的双层;通过用拉曼光谱技术补充AFM，确定蛋白质的存在或不存在，并给出与相互作用相关的构象/结构变化的指示;并进一步发展了原子力显微镜技术，用于测量一般双层膜的基本热力学参数。

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.