Electrostatic potentials and biological membranes

描述(由申请人提供)：长期目标是了解物理因素(静电学、扩散、降维)如何通过钙/磷肌醇第二信使系统产生信息流。磷脂酰肌醇4，5-二磷酸，PIP2，是三个第二信使的来源，并参与广泛的膜相关现象，如离子通道或酶的激活，内吞和胞吐。PIP2是如何做到这一点的？工作假设是Marcks和其他蛋白质上的无结构碱性/疏水残基簇充当可逆的PIP2缓冲区：它们产生局部正静电势，隔离多价酸性脂类。当局部钙离子水平升高时，钙/钙调蛋白(Ca/CaM)与簇结合，释放PIP2。六个具体目标将探索这一假说。首先，一个简单的着色方案和多肽结合测量将被用来识别医学上重要蛋白质上的基本/疏水簇。这种方法使PI能够识别受体酪氨酸激酶(RTK)、G蛋白偶联受体和支架蛋白上的感兴趣区域，这些蛋白在癌症和药物靶标中具有重要作用。PC、FRET、荧光阻流和其他测量将用于确定膜、Ca/CaM和PIP2的多肽亲和力。这些相互作用的生物学后果将与细胞生物学家合作进行研究。最近在Gravin上发现了三个这样的簇，这说明了生物物理/细胞生物学相结合的方法的力量。第二，利用荧光测量来确定钙/钙调素与重组肽结合的动力学和寿命，这些重组肽对应于固有膜蛋白的碱性/疏水近膜+跨膜区。第三，确定非结构碱性簇如何为PIP2提供足够的特异性，以靶向质膜上的蛋白质，如精子因子PLcheeta。第四，验证GAP-43/神经调制素在神经元轴突生长锥体中作为可逆的PIP2受体的假设。第五，为了证明碱性/疏水性多肽在结合到缺乏PIP2的膜上时比脂类扩散得更快，防止了磷脂酰丝氨酸和胆固醇等其他脂类的滞留。第六，测试蛋白质的碱性/疏水区(如Marcks、Gravin、RTKs)如何形成富含胆固醇的“筏”的模型。

DESCRIPTION (provided by applicant): The long term objective is to understand how physical factors (electrostatics, diffusion, reduction of dimensionality) produce a flow of information through calcium/phosphoinositide second messenger systems. Phosphatidylinositol 4,5-bisphosphate, PIP2, is the source of three second messengers and is involved in a wide range of membrane-related phenomena, such as the activation of ion channels or enzymes, endocytosis, and exocytosis. How does PIP2 do so much? The working hypothesis is that unstructured clusters of basic/hydrophobic residues on MARCKS and other proteins act as reversible PIP2 buffers: they produce a local positive electrostatic potential that sequesters the polyvalent acidic lipid. Upon a local increase in the level of Ca2+, calcium/calmodulin (Ca/CaM) binds to the cluster, releasing the PIP2. Six specific aims will explore this hypothesis. First, a simple coloring scheme and peptide binding measurements will be used to identify basic/hydrophobic clusters on medically important proteins. This approach has allowed the PI to identify regions of interest on receptor tyrosine kinases (RTK), G protein coupled receptors, and scaffolding proteins that are important in cancer and as drug targets. PCS, FRET, fluorescence stop flow, and other measurements will be used to determine peptide affinity for membranes, Ca/CaM, and PIP2. The biological consequences of these interactions will be investigated in collaborations with cell biologists. The recent identification of three such clusters on gravin illustrates the power of the combined biophysical/cell biology approach. Second, to use fluorescence measurements to determine the kinetics and life time of Ca/CaM binding to reconstituted peptides corresponding to the basic/hydrophobic juxtamembrane + transmembrane regions of intrinsic membrane proteins. Third, to determine how unstructured basic clusters provide sufficient specificity for PIP2 to target proteins such as the sperm factor PLCzeta to the plasma membrane. Fourth, to test the postulate that GAP-43/neuromodulin acts as a reversible PIP2 sink in the axonal growth cones of neurons. Fifth, to show that the basic/hydrophobic peptides diffuse more rapidly than lipids when bound to membranes that lack PIP2, preventing sequestration of other lipids such as phosphatidylserine and cholesterol. Sixth, to test a model for how basic/hydrophobic regions of proteins (e.g. MARCKS, gravin, RTKs) may nucleate the formation of cholesterol-enriched "rafts".