ELECTROSTATIC POTENTIALS AND BIOLOGICAL MEMBRANES

描述：长期目标是了解物理因素如何 例如静电学和降维产生了 通过钙/磷脂第二信使系统传递信息。这 提案的重点是Marcks，一种普遍存在的蛋白激酶C的主要底物 (PKC)可逆地与钙调蛋白、肌动蛋白和膜结合。二 假说将会得到检验。第一个假设是，膜结合 Marcks需要将其N-末端肉豆蔻酸插入到内部 碱性残基的双层结构及其与酸性物质的相互作用 脂类。丝氨酸残基的PKC磷酸化减弱了静电 相互作用并产生Marcks从膜到细胞质的转位。 第二个假设是马克可以自发地自组装成 具有单价酸性脂类的侧向结构域；另一种不太常见 此第二信使系统的组件(例如PKC、Src和PIP2)将 因为静电的考虑而加入域；并且这些 信号转导结构域具有重要的生理功能。这个 三个具体目标是通过确定Marcks如何 结合到膜上，它是如何与酸性脂质形成结构域的，以及 这些领域的意义。使用原子模型的理论计算 的膜和多肽将与实验测量相结合 实现这些目标。活性、荧光和膜的测量 结合将使用重组的信号转导系统来执行 由磷脂小泡和纯化的蛋白质组成(异三聚体G 蛋白质亚基、磷脂酶C、蛋白激酶C、血源蛋白和标记)。Marcks是 医学上很重要，因为它与分泌、细胞 运动，调节细胞周期和转化。这些研究还包括 与其他医学上重要的蛋白质有关，这些蛋白质使用 与酸性磷脂结合的碱性残基，例如K-RAS 4B、Src和 HIV-1基质蛋白。最后，初步数据表明，区域形成了 由Marcks和酸性脂质PIP2产生的一种新的信号放大 基于正反馈概念的转导导致“全部或全部” 无“回答。

DESCRIPTION: The long term objective is to understand how physical factors such as electrostatics and reduction of dimensionality produce a flow of information through the calcium/phospholipid second messenger system. This proposal focuses on MARCKS, a ubiquitous major substrate of protein kinase C (PKC) that binds reversibly to calmodulin, actin, and membranes. Two hypotheses will be tested. The first hypothesis is that membrane binding of MARCKS requires both insertion of its N-terminal myristate into the interior of the bilayer and interaction of its cluster of basic residues with acidic lipids. PKC phosphorylation of serine residues weakens the electrostatic interaction and produces translocation of MARCKS from membrane to cytoplasm. The second hypothesis is that MARCKS can spontaneously self assemble into lateral domains with monovalent acidic lipids; that other, less prevalent components of this second messenger system (e.g. PKC, Src, and PIP2) will join the domains because of electrostatic considerations; and that these signal transduction domains have important physiological functions. The three specific aims are to test these hypotheses by determining how MARCKS binds to membranes, how it forms domains with acidic lipids, and the significance of these domains. Theoretical calculations using atomic models of membranes and peptides will be combined with experimental measurements to achieve these aims. Measurements of activity, fluorescence, and membrane binding will be performed using a reconstituted signal transduction system composed of phospholipid vesicles and purified proteins (heterotrimeric G protein subunits, phospholipase C, PKC, Src, and MARCKS). MARCKS is medically important because it has been implicated in secretion, cell motility, regulation of the cell cycle and transformation. The studies also are relevant to other medically important proteins that use clusters of basic residues to bind to acidic phospholipids, e.g. K-Ras 4B, Src, and HIV-1 matrix protein. Finally, preliminary data suggest that domains formed by MARCKS and the acidic lipid PIP2 produce a novel amplification of signal transduction based on the concept of positive feedback leading to an "all or none" response.