SINGLE MOLECULE ENZYMOLOGY: A FIELD/TRAP APPARATUS

长期目标是开发用于测量单个酶分子活性的技术和仪器，这些酶分子可以改变其膜结合底物的电荷，例如脂肪酶、激酶和磷酸酶。结合微电泳和激光陷阱技术，现场/陷阱装置采用密立根油滴实验的原理：涂有磷脂双分子层的硅珠代替油滴，紧密聚焦的激光束代替重力。当在盐溶液中施加交流电场时，电泳力将其从激光阱中的平衡位置移位。用快速四象限二极管测量的位移与头部外小叶上带电脂质的数量成正比（例如磷脂酰肌醇，4,5-二磷酸，IPI2）。当含有酶（如磷脂酶C， PLC）的溶液流过珠粒时，蛋白质吸附到表面并改变珠粒上的电荷（如水解三价PIP2形成中性脂质二酰基甘油）。构造了一个原型装置来演示原理验证，并用于PLC-delta的研究。具体目标1是在Stony Brook建立一个新的设备，能够通过单个PLC检测10-100个PIP2的水解，最初含有10,000个PIP2，时间分辨率为0.1-1.0秒。具体目标2是通过增加荧光相关光谱（fcs）能力来扩展场/陷阱方法，这将能够同时测量单个酶的荧光信号及其活性。现场/陷阱方法将应用于研究具有重要生物学和医学意义的酶：被G蛋白激活时产生两个第二信使的plc - β同工异构体；脂质激酶PI3K，它产生另一类与癌症有关的第二信使；PTEN是一种脂质磷酸酶，是一种高度突变的临床重要肿瘤抑制因子。

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.