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.
