Electrostatics Mediates the Membrane Binding of Src, a Myristoylated Protein

总的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v-src及其细胞同源物c-Src的产物，统称为Src)；工作假设是，膜结合既需要nh2末端肉豆酸酯疏水插入到双层中，也需要一簇邻近的碱性残基与酸性脂质的静电相互作用。有四个具体的项目。1)建立了一个简单的模型来描述肉豆蔻酸酯和碱性残基对Src与磷脂膜结合的协同作用，并通过测量Src的nh2末端区域对应的肉豆蔻酸酯化肽和非肉豆蔻酸化肽的膜结合来验证该模型。2)通过测量的膜结合来证实Src的nh2末端区域的碱性残基负责蛋白-膜相互作用的静电成分原生v-Src和c-Src以及这些蛋白质的突变和嵌合版本。3)测定C和A激酶磷酸化对Src及相应肽的膜结合的影响。4)构建磷脂双分子层和Src的nh2末端区域的分子模型，并利用非线性泊松-玻尔兹曼方程从理论上计算了这种结合的静电分量。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豆芽糖酰化（如MARCKS）和法酰化（如K-Ras）蛋白质。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，其功能是转化m细胞，其细胞同源物，c-Src，参与控制细胞周期，统称为Src)；工作假设是，膜结合既需要nh2末端肉豆酸酯疏水插入到双层中，也需要一簇邻近的碱性残基与酸性脂质的静电相互作用。这是四个具体的项目。1)建立一个简单的模型，描述肉豆酸酯和碱性残基对Src与磷脂膜结合的协同作用，并通过测量合成肽的膜结合来验证该模型。2)通过测定天然v-Src和c-Src的膜结合以及突变型和嵌合型，证实Src nh2末端区域的碱性残基负责蛋白-膜相互作用的静电组分。3)测定C和A激酶磷酸化对Src及相应肽的膜结合的影响。4)构建磷脂双分子层和Src的nh2末端区域的分子模型，并从理论上计算其结合的静电分量。该项目意义重大，因为膜结合对Src的功能至关重要，并且所出现的生物物理原理应该适用于其他肉豆蔻酰基化蛋白。* * *

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. ***