Electrostatics and Lateral Domains in Membranes

目前的假设是，物理因素（如静电、表面压力）可导致特定脂质和蛋白质自组装到细胞膜的侧结构域，这些结构域对细胞信号传导很重要。具体目标是了解(1)基本肽在磷脂囊泡中形成侧结构域的分子机制；（ii）细胞膜中高浓度存在的蛋白质（如小窝蛋白）如何自组装成结构域；（iii）其他细胞信号蛋白（如Src）和脂质（如磷脂酰肌醇4,5，-二磷酸（PIP2））如何被隔离在这些结构域中。理论和实验将互动发展，以提供基本的生物物理信息，这一复杂而重要的问题在信号转导。将使用荧光、表面压力和其他技术研究由钙/磷脂第二信使系统（例如PIP2、PKC、Src、G蛋白、磷脂酶C）和小窝蛋白支架区（残基82-101）对应的肽组成的重组系统。理论计算将使用肽，蛋白质和膜的现实分子模型进行。四个可能有助于结构域形成的因素将被检查：蛋白质或肽的自聚集；W、F、Y残基对极头基区域的穿透；Onsager描述的熵长杆效应；和静电学。将制定解释每个因素对畴形成的相对贡献的理论。初步结果和计算表明，即使是简单的碱性肽，如Lys5和小泡蛋白的支架结构域，也可以通过非特异性静电相互作用将PIP2隔离在侧结构域，这可能为小泡中这种脂质的隔离提供了分子解释。这个生物物理学项目具有直接的生物学相关性，因为有充分的证据表明，生物膜包含几种不同类型的侧向结构域，这些结构域在信号转导中很重要。例如，称为小窝的质膜侧结构域含有高浓度的小窝蛋白和信号分子，如异三聚体G蛋白、Src家族激酶、PKC和PIP2；后者是一种重要的脂质，是两种第二信使的来源，并将几种蛋白质锚定在膜上。在受体刺激下被水解的PIP2大部分位于小泡中，这表明它们可能是激动剂刺激下PIP2转换的主要部位。＊＊＊

9729538 McLaughlin The working hypothesis is that physical factors (e.g. electrostatics, surface pressure) can lead to the self assembly of specific lipids and proteins into lateral domains in cell membranes and that these domains are important for cell signaling. The specific objectives are to understand (1) the molecular mechanisms by which basic peptides form lateral domains in phospholipid vesicles; (ii) how proteins present at high concentrations in the cell membrane, e.g. caveolin, self assemble into domains; and (iii) how other cell signaling proteins, e.g. Src, and lipids, e.g. phosphatidylinositol 4,5,-bisphosphate (PIP2), are sequestered in these domains. Theory and experiment will be developed interactively to provide fundamental biophysical information about this complicated but important problem in signal transduction. Reconstituted systems comprising components of the calcium/phospholipid second messenger system (e.g. PIP2, PKC, Src, G proteins, phospholipase C) and peptides corresponding to the scaffolding region of caveolin (residues 82-101) will be studied using fluorescence, surface pressure, and other techniques. Theoretical calculations will be performed using realistic molecular models of peptides, proteins, and membranes. Four factors that may contribute to domain formation will be examined: self-aggregation of the proteins or peptides; penetration of the polar head group region by W, F, and Y residues; the entropic long rod effect described by Onsager; and electrostatics. Theories that account for each factor's relative contribution to domain formation will be formulated. Preliminary results and calculations show that even simple basic peptides such Lys5 and the scaffolding domain of caveolin can sequester PIP2 in lateral domains via nonspecific electrostatic interactions, which may provide a molecular explanation for the sequestration of this lipid in caveolae. This biophysics project has direct biological relevance because there is good evidence that biologic al membranes contain several different types of lateral domains that are important in signal transduction. For example, the plasma membrane lateral domains called caveolae contain high concentrations of caveolin and signaling molecules such as heterotrimeric G proteins, Src family kinases, PKC, and PIP2; the latter is an important lipid that is both the source of two second messengers and acts to anchor several proteins to membranes. Much of the PIP2 that is hydrolyzed in response to receptor stimulation is localized in the caveolae, suggesting they may be a primary site of agonist-stimulated PIP2 turnover. ***