Electrostatics Mediates the Membrane Binding of Src, a Myristoylated Protein

9419175 McLaughlin总体目标是了解含有肉豆蔻酸酯（一种14碳酰基链）的蛋白质与生物膜相互作用的分子机制。 该提案的重点是Src（v-src癌基因的产物v-Src及其细胞同系物c-Src，统称为Src）;工作假设是膜结合需要将NH 2-末端肉豆蔻酸酯疏水插入到双层中，以及相邻碱性残基簇与酸性脂质的静电相互作用。 有四个具体项目。1)开发一个简单的模型，描述了协同作用的肉豆蔻酸酯和碱性残基的Src磷脂膜的结合和测试该模型通过测量膜结合的肉豆蔻酰化和nonmyristoylated肽对应的NH 2-末端区域的Src。2)通过测量天然v-Src和c-Src以及这些蛋白质的突变体和嵌合体的膜结合，证实Src的NH 2-末端区域中的碱性残基负责蛋白质-膜相互作用的静电组分。3)测量C和A激酶磷酸化对Src和相应肽两者的膜结合的影响。4)构建磷脂双层膜和Src氨基端区域的分子模型，并利用非线性Poisson-Boltzmann方程从理论上计算了结合的静电分量。 该项目意义重大，因为膜结合对Src的功能至关重要。 出现的生物物理原理应该适用于其他一些肉豆蔻酰化（如MARCKS）和法尼基化（如K-Ras）蛋白。 总体目标是了解含有肉豆蔻酸酯（一种14碳酰基链）的蛋白质与生物膜相互作用的分子机制。 该提案侧重于Src（v-src癌基因的产物v-Src，其作用是转化细胞，其细胞同系物c-Src，其参与细胞周期的控制，统称为Src）;工作假设是膜结合需要NH 2-的疏水插入，末端肉豆蔻酸酯进入双分子层和一簇相邻的碱性残基与酸性脂质的静电相互作用。 这是四个具体项目。1)开发一个简单的模型，描述了协同作用的肉豆蔻酸酯和碱性残基的结合Src磷脂膜和测试这个模型通过测量合成肽的膜结合。2)通过测量天然v-Src和c-Src以及这些蛋白质的突变体和嵌合体的膜结合，证实Src的NH 2-末端区域中的碱性残基负责蛋白质-膜相互作用的静电组分。3)测量C和A激酶磷酸化对Src和相应肽两者的膜结合的影响。4)构建了磷脂双分子层与Src氨基端结合的分子模型，并从理论上计算了结合的静电分量。 该项目意义重大，因为膜结合对Src的功能至关重要，并且出现的生物物理原理应该适用于其他肉豆蔻酰化蛋白质。 ***

9419175 McLaughlin The overall objective is to understand the molecular mechanism by which proteins containing myristate, a 14 carbon acyl chain, interact with biological membranes. The proposal focuses on Src (the product of the v-src oncogene, v-Src, and its cellular homolog, c-Src, are known collectively as Src); the working hypothesis is that membrane binding requires both the hydrophobic insertion of the NH2-terminal myristate into the bilayer and the electrostatic interaction of a cluster of adjacent basic residues with acidic lipids. There are four specific projects. 1) Developing a simple model that describes the synergistic effect of myristate and basic residues on the binding of Src to phospholipid membranes and testing this model by measuring the membrane binding of myristoylated and nonmyristoylated peptides corresponding to the NH2-terminal region of Src. 2) Confirming that the basic residues in the NH2-terminal region of Src are responsible for the electrostatic component of protein-membrane interaction by measuring the membrane binding of native v-Src and c-Src as well as mutant and chimeric versions of these proteins. 3) Measuring the effect of phosphorylation by C and A kinases on the membrane binding of both Src and corresponding peptides. 4) Constructing a molecular model of a phospholipid bilayer and the NH2-terminal region of Src, then using the nonlinear Poisson-Boltzmann equation to calculate theoretically the electrostatic component of the binding. The project is significant because membrane binding is crucial to the function of Src. The biophysical principles that emerge should be applicable to some other myristoylated (e.g. MARCKS) and farnesylated (e.g. K-Ras) proteins. %%% The overall objective is to understand the molecular mechanism by which proteins containing myristate, a 14 carbon acyl chain, interact with biological membranes. The proposal focuses on Src (the product of the v-src oncogene, v-Src, which functions to transfor m cells, and its cellular homolog, c-Src, which is involved in control of the cell cycle, are known collectively as Src); the working hypothesis is that membrane binding requires both the hydrophobic insertion of the NH2-terminal myristate into the bilayer and the electrostatic interaction of a cluster of adjacent basic residues with acidic lipids. These are four specific projects. 1) Developing a simple model that describes the synergistic effect of myristate and basic residues on the binding of Src to phospholipid membranes and testing this model by measuring the membrane binding of synthetic peptides. 2) Confirming that the basic residues in the NH2-terminal region of Src are responsible for the electrostatic component of protein-membrane interaction by measuring the membrane binding of native v-Src and c-Src as well as mutant and chimeric versions of these proteins. 3) Measuring the effect of phosphorylation by C and A kinases on the membrane binding of both Src and corresponding peptides. 4) Constructing a molecular model of a phospholipid bilayer and the NH2-terminal region of Src, then calculating theoretically the electrostatic component of the binding. The project is significant because membrane binding is crucial to the function of Src and the biophysical principles that emerge should be applicable to other myristoylated proteins. ***