Phospholipid dynamics and lipolysis in membrane models

说明(申请人提供)：磷脂在生物膜的物理化学性质中起着关键作用。由于膜和膜介导的生物过程中脂-脂和脂-蛋白相互作用的动态特征，表征磷脂在自组装和对添加的代谢物(如其他膜成分)的响应中的结构和动力学是理解膜生物物理学的重要一步，因为它与细胞膜功能和信号转导有关。这项研究旨在利用一系列新设计的双标记磷脂类似物和定位(定点)荧光报告基团作为膜模型来表征磷脂在囊泡双层和胶束中的组织、结构和动力学行为。将在(I)两个脂肪酰基链末端的每一个处引入光谱标记，以表征链-链相互作用，以及(Ii)在头基和每个酰基链上每次引入一个光谱标记，以确定极性头基和每个疏水尾部之间的距离/取向。这些探针将被结合到具有特定组成和物理化学参数的囊泡和胶束中。荧光共振能量转移(FRET)的测量将被用来表征探针。提出的假设是，确定这些合成类似物中磷脂分子组成荧光基团之间的距离和方向将为确定膜中磷脂行为的结构和动力学提供一种新的技术。通常使用的双标记荧光磷脂类似物几乎只用于通过监测分子内荧光猝灭的损失来实时光谱分析磷脂酶，而本文介绍的光谱探针被设计为真正的“光谱尺子”，通过直接测量标记之间的FRET效率来确定磷脂分子中荧光基团的距离和方向。阐明膜中磷脂的结构和动力学，以及它们与其他膜脂和蛋白质相互作用的方式，将为更好地理解膜在健康和疾病中的行为提供重要信息。 与公共卫生相关：膜在生物过程中发挥重要作用；它们参与通讯、细胞信号、炎症和过敏。磷脂是所有生物膜的关键成分，对膜的结构和功能起着重要的作用。阐明膜上的磷脂结构以及它们与其他膜脂和蛋白质相互作用的方式将为更好地理解膜在健康和疾病中的行为提供有用的信息。

DESCRIPTION (provided by applicant): Phospholipids have a key role in contributing to the physicochemical properties of biological membranes. Because of the dynamic character of lipid-lipid and lipid-protein interactions in membranes and membrane mediated biological processes, characterization of the structure and dynamics of phospholipids in self-assembly and in response to addition of metabolites (e.g., other membrane components) is an important step toward understanding membrane biophysics as it relates to cell membrane function and signal transduction. The proposed research aims at characterization of the organization, structure and dynamic behavior of phospholipids in vesicle bilayers and micelles as membrane models using a series of newly designed double- labeled phospholipid analogues with strategically placed (site-directed) fluorescent reporter groups. The spectroscopic labels will be introduced (i) at each of the two fatty acyl chain-ends to characterize chain-chain interactions, and (ii) at the headgroup and each acyl chain, one at a time, to determine the distance / orientation between the polar headgroup and each one of the hydrophobic tails. These probes will be incorporated into vesicles and micelles of defined compositions and physicochemical parameters. Measurements of fluorescence resonance energy transfer (FRET) will be used to characterize the probes. The proposed hypothesis is that determination of the distance and orientation between the constituent fluorescent groups of phospholipid molecules in these synthetic analogues will provide a new technology to determine the structure and dynamics of phospholipid behavior in membranes. In contrast to the commonly used double- labeled fluorescent phospholipid analogues that are almost exclusively used for real-time spectroscopic assay of phospholipases by monitoring the loss of intramolecular fluorescence quenching, the spectroscopic probes here introduced are designed to be used as true "spectroscopic rulers" to determine the distance and orientation of the fluorescent groups in the phospholipid molecule by direct measurement of the FRET efficiency between the labels. Elucidation of phospholipid structure and dynamics in membranes and the way they interact with other membrane lipids and proteins will provide important information toward better understanding membrane behavior in health and disease. PUBLIC HEALTH RELEVANCE: Membranes play an important role in biological processes; they are involved in communication, cell signaling, inflammation, and allergy. Phospholipids are key components in all biological membranes and they contribute to membrane structure and function. Elucidation of phospholipid structure in membranes and the way they interact with other membrane lipids and proteins will provide useful information toward better understanding of membrane behavior in health and disease.