SINGLE MOLECULE ENZYMOLOGY: A FIELD/TRAP APPARATUS

单分子酶学：场/陷阱装置

• 批准号: 6729157
• 负责人: Stuart G McLaughlin
• 金额: $ 18.53万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6729157
• 关键词: analytical method; bioengineering /biomedical engineering; biomedical equipment development; biophysics; chemical kinetics; computer data analysis; electric field; electrophoresis; enzyme activity; enzyme induction /repression; enzyme mechanism; enzyme substrate; fluorescence spectrometry; immobilized enzymes; lasers; mathematics; nanotechnology; phosphatidylinositols; phospholipase C; silicates; time resolved data

The long-term objective is to develop technology and instrumentation for measuring the activity of single enzyme molecules that change the charge of their membrane-bound substrates, e.g. lipases, kinases and phosphatases. Combining microelectrophoresis and laser trap technologies, the field/trap apparatus uses the principle of the Millikan oil drop experiment: a silica bead coated with phospholipid bilayer replaces the oil drop and tightly focused laser beam replaces gravity. When an AC field is applied to the coated bead in a salt solution, the electrophoretic force displaces it from its equilibrium position in the laser trap. The displacement, measured with a fast quadrant diode is a proportional to the number of charged lipids (e.g. phosphatidylinositol, 4,5-biphosphate, IPI2) on the outer leaflet of the bead. When a solution containing enzyme (e.g. phospholipase C, PLC) flows past the bead, the proteins adsorb to the surface and change the charge on the bead (e.g. hydrolyze trivalent PIP2 to form the neutral lipid diacylglycerol). A prototype apparatus has been constructed to demonstrate proof-of- principle and used to study PLC-delta. Specific aim 1 is to construct a new apparatus at Stony Brook that will be capable of detecting hydrolysis of 10-100 PIP2 by a single PLC on a bead, initially containing 10,000 PIP2, with a time resolution of 0.1-1.0 sec. Specific Aim 2 is to expand the field/trap approach by adding fluorescence correlation spectroscopy (fcs) capability, which will enable simultaneous measurement of the fluorescence signal from a single enzyme and its activity. The field/trap approach will be applied to study enzymes of great biological and medical importance: PLC-beta isoforms that produce two second messengers when activated by G proteins; the lipid kinase PI3K, which produces another class of second messengers that have been implicated in cancer; and PTEN, a lipid phosphatase that is a highly mutated clinically important tumor suppressor.

长期目标是开发技术和仪器来测量单个酶分子的活性，这些酶分子会改变其膜结合底物的电荷，例如脂肪酶、激酶和磷酸酶。该场/陷阱装置结合了微电泳和激光捕获技术，采用了密立根油滴实验的原理：涂有磷脂双层的硅珠代替了油滴，紧密聚焦的激光束代替了重力。当交流场施加到盐溶液中的涂层珠上时，电泳力将其从激光阱中的平衡位置移开。使用快速象限二极管测量的位移与珠子外层的带电脂质（例如磷脂酰肌醇、4,5-二磷酸盐、IPI2）的数量成正比。当含有酶（例如磷脂酶 C、PLC）的溶液流过珠子时，蛋白质吸附到表面并改变珠子上的电荷（例如水解三价 PIP2 形成中性脂质二酰基甘油）。已经构建了原型设备来演示原理验证并用于研究 PLC-delta。具体目标 1 是在石溪建造一个新装置，能够通过单个 PLC 在珠子上检测 10-100 PIP2 的水解，最初包含 10,000 PIP2，时间分辨率为 0.1-1.0 秒。具体目标 2 是通过添加荧光相关光谱 (fcs) 功能来扩展场/陷阱方法，这将能够同时测量单个酶的荧光信号及其活性。场/陷阱方法将用于研究具有重大生物学和医学重要性的酶：被 G 蛋白激活时产生两个第二信使的 PLC-β 亚型；脂质激酶 PI3K，它产生另一类与癌症有关的第二信使； PTEN，一种脂质磷酸酶，是一种高度突变的临床重要肿瘤抑制因子。