Matthew Beyersdorf

University Honors with Honors in Chemistry

Major: Chemistry

Supervisor: Christopher Halkides, Chemistry and Biochemistry

Analysis, Purification, and Crystallization of Thermatoga maritima Phosphono-CheY

An analog of a signal transduction protein, Thermatoga maritima CheY, was produced, purified, and allowed to undergo an alkylation reaction with phosponomethyltriflate to give rise to phosphono-CheY. This newly generated protein is a stable analog of CheY-phosphate, a protein which has a half-life of less than a minute in vivo. Phosphono-CheY was studied because of the ability of CheY to assist with the regulation of bacterial chemotaxis. Our modified protein was analyzed, purified, and concentrated using methods of reverse phase HPLC and cation exchange HPLC. Upon obtaining pure Phosphono-CheY, the protein was crystallized using a hanging drop method over a wide array of conditions. Ultimately, two crystals were able to be diffracted using a high energy synchrotron source. These crystals which gave reasonable diffraction patterns and will allow for analysis into the atomic structure of the protein were crystallized under the following conditions: 24% PEG 6000, 20 mM MgCl2, 100 mM HEPES buffer (pH 7.0), and 150 mM (NH4)2SO4; 26% PEG 3400, 15 mM MgCl2, 100 mM HEPES (pH 7.0), and 200 mM (NH4)2SO4. These two crystals diffracted to 1.2 Å and 1.5 Å respectively. Further research to be done includes using the collected diffraction pattern data sets to refine the atomic structure of phosphono-CheY and the co-crystallization of Phosphono-CheY with other proteins involved in the chemotaxis of Thermatoga maritima.