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、扩散、降维）如何通过钙/磷酸肌苷第二信使系统产生信息流。磷脂酰肌醇4,5-二磷酸（PIP2）是三秒信使的来源，并参与广泛的膜相关现象，如离子通道或酶的激活、内吞作用和胞吐作用。PIP2是如何发挥这么大作用的？工作假设是，MARCKS和其他蛋白质上的碱性/疏水性残基的非结构化簇作为可逆的PIP2缓冲液：它们产生局部正静电电位，隔离多价酸性脂质。当局部Ca2+水平升高时，钙/钙调蛋白（Ca/CaM）结合到簇上，释放PIP2。六个具体目标将探讨这一假设。首先，简单的着色方案和肽结合测量将用于识别医学上重要蛋白质上的碱性/疏水性簇。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，这些区域在癌症和药物靶点中很重要。PCS， FRET，荧光停止流和其他测量将用于确定肽对膜，Ca/CaM和PIP2的亲和力。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，说明了生物物理/细胞生物学结合方法的力量。其次，利用荧光测量来确定Ca/CaM与内在膜蛋白的碱性/疏水性近膜+跨膜区域对应的重构肽结合的动力学和寿命。第三，确定非结构化的基本簇如何为PIP2提供足够的特异性，使精子因子PLCzeta等蛋白靶向质膜。第四，验证GAP-43/神经调节素在神经元轴突生长锥中作为可逆PIP2汇的假设。第五，当碱性/疏水肽结合到缺乏PIP2的膜上时，比脂质扩散得更快，从而阻止了磷脂酰丝氨酸和胆固醇等其他脂质的隔离。第六，测试蛋白质的碱性/疏水性区域（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".