ELECTROSTATIC POTENTIALS AND BIOLOGICAL MEMBRANES

长期目标是了解物理因素，如 静电势和降维有助于产生 信息在钙/磷脂中的时空流动 第二信使系统。这第二种蛋白质的三个主要成分 信号转导系统--磷脂酶C、蛋白激酶C及其主要 底物Marcks蛋白质-所有包含碱性残基的簇 似乎与细胞中的酸性磷脂短暂地相互作用 在这个系统的激活过程中的膜。下一步的主要目标 五年是为了了解蛋白质上的碱性残基簇是如何结合的 通过研究这三种蛋白质、多肽对膜中酸性脂类的影响 模拟这些蛋白质的相关碱性区域，并简单地 合成肽。这个生物物理项目与健康有关： 已经提出了定量的可证伪模型来解释为什么肿瘤 促进佛波酯与酸性磷脂协同作用 激活PKC，以及为什么一簇碱性残基与 Marcks(和相关的神经调素)蛋白对酸性磷脂的作用 质膜是丝氨酸蛋白激酶C磷酸化过程中不可或缺的一步 这些簇内的残基和伴随的局部释放 钙调素结合到这些簇上。小的合成碱性多肽将是 用于确定与磷脂小泡的结合如何依赖于 基本残留物的数量、分布和化学性质，以及 温度和压力。支付的“熵价格”的大小 在绑定时也将进行测量。过于简单化的古伊- 静电脂-肽相互作用的查普曼/质量作用模型 通过结合现有的衍射和2H-核磁共振数据来改进 将所述极性头基定向为膜的分子模型， 然后将真实的部分电荷分配给这些头基团上的原子 用三维Poisson-Boltzmann方程计算 静电势。关于多肽的结构信息是必需的 对于这个新模型，将使用圆二色谱和二维核磁共振在 与D.Cafiso(弗吉尼亚州大学)合作。细胞的膜结合作用 磷酸化和非磷酸化形式的25个氨基酸的多肽， 包含PKC磷酸化位点和钙调蛋白结合域 将研究Marcks，以及该区域对完好的 蛋白。使用的技术包括过滤、平衡透析、 载蔗糖小泡的离心法，凝胶迁移率， 量热法、单层压力和荧光光谱分析。

The long term objective is to understand how physical factors such as electrostatic potentials and reduction of dimensionality help to produce a spatial and temporal flow of information through the calcium/phospholipid second messenger system. The three major protein components of this second messenger system - phospholipase C, protein kinase C (PKC), and its major substrate the MARCKS protein - all contain clusters of basic residues that appear to interact transiently with acidic phospholipids in the cell membrane during activation of this system. The main objective for the next five years is to understand how clusters of basic residues on proteins bind to acidic lipids in membranes by studying these three proteins, peptides that mimic the relevant basic regions of these proteins and simple synthetic peptides. This biophysical project is health related: quantitative falsifiable models have been proposed to explain why the tumor promoting phorbol esters act synergistically with acidic phospholipids to activate PKC, and why the binding of a cluster of basic residues on the MARCKS (and related neuromodulin) protein to acidic phospholipids in the plasma membrane is an integral step in both the PKC phosphorylation of Ser residues within these clusters and the concomitant localized release of calmodulin bound to these clusters. Small synthetic basic peptides will be used to determine how the binding to phospholipid vesicles depends on the number, distribution, and chemical nature of the basic residues, as well as the temperature and pressure. The magnitude of the "entropy price" paid upon binding also will be measured. the highly oversimplified Gouy- Chapman/mass action model for electrostatic lipid-peptide interactions will be improved by incorporating existing diffraction and 2H-NMR data about the orientation of the polar head groups into a molecular model of a membrane, then assigning realistic partial charges to atoms on these head groups and using the three dimensional Poisson-Boltzmann equation to calculate the electrostatic potential. Structural information about peptides necessary for this new model will be obtained using circular dichroism and 2D NMR in collaboration with D. Cafiso (U. Virginia). The membrane binding of the phosphorylated and unphosphorylated forms of a 25-amino acid peptide that contains the PKC phosphorylation sites and calmodulin binding domain of MARCKS will be studied, as will the binding of this region on the intact protein. Techniques to be used include filtration, equilibrium dialysis, centrifugation of sucrose-loaded vesicles, electrophoretic mobility, calorimetry, monolayer pressure, and fluorescence spectroscopy.