This award to San Jose State University will provide funding for purchase of a specialized instrument that detects the movement of fluorescent molecules when introduced to a small localized temperature gradient, a process known as thermophoresis. Because the physical properties of a molecule change when bound to another molecule, thermophoresis experiments can be used to measure the binding affinity of two interacting partners, as long as one of the molecules is fluorescent or labeled with a fluorescent probe. Initial experiments will focus on three projects by three different investigators. The Skovran laboratory will use thermophoresis to characterize the expression and regulation of the key enzymes that allow microorganisms to utilize methane or methanol as a carbon source; this project could enhance metabolic engineering efforts that aim to design microorganisms for the production of biofuels and other desired chemicals. The Rascon laboratory will use thermophoresis to study certain digestive enzymes from mosquitos that bind and cleave blood-meal proteins; this project could lead to new strategies of controlling mosquito populations that carry serious diseases like the Dengue fever pathogen. The Eggers laboratory plans to use thermophoresis to test a new theory regarding the role of water in binding reactions; results from this project may be transformative for the field of biothermodynamics. Numerous undergraduate and MS/MA students will be trained in operation of the thermophoresis instrument, including minority students supported by other programs at SJSU.
The microscale thermophoresis (MST) instrument to be purchased with this award employs a fluorescence detector with a built-in infrared laser that heats a nanoliter-sized volume of sample at the exact same location where fluorescence emission is monitored. The phenomenon of thermophoresis refers to the migration of species into/out of the heated zone due to differences in molecular size, surface chemistry, and hydration properties. If one of two binding species is fluorescently labeled, MST may be used to separate and measure the bound complex relative to the free reactants, yielding an equilibrium binding constant. Initial MST experiments will focus on three projects: (1) characterization of the key binding events that lead to expression and regulation of methanol dehydrogenases in a model organism, Methylobacterium extorquens; (2) the investigation of proteases from the mosquito Aedes aegypti to test the hypothesis that enzyme expression and activity are organized in a proteolytic cascade to optimize blood-meal protein digestion; and (3) the study of model enzyme-inhibitor interactions to test a new thermodynamic framework that incorporates the role of desolvation in the governing equations for binding equilibria. The use of microscale thermophoresis in these projects has several advantages over other binding techniques, including low reagent consumption, an unrestricted range of operating concentrations, quick analysis time, and the potential to analyze macromolecules in cell lysates without prior purification.