Maxwell Darch

Honors in Chemistry

Major: Chemistry

Supervisor: Chris Halkides, Chemistry and Biochemistry

Optimization of Crystallization Conditions for phosphono-CheY, a Stable Analog of in vivo CheY-phosphate

A stable analog form of a response regulator, CheY protein, in Thermotoga maritima was synthesized, purified, and modified for optimizing crystallization conditions. This protein is a response regulator involved in the bacterial chemotaxis signal transduction pathway. The in vivo form of CheY is phosphorylated at a critical aspartate residue that activates the protein allowing it to bind the flagellar motor affecting the rotational direction of the flagellum. This process allows the bacterium to move toward more favorable locations in its environment (chemotaxis). The phosphorylated form is very short lived; therefore, the aspartate residue is mutated to a cysteine residue at position 54. This mutation is then subject to alkylation resulting in the addition of a phosphonomethyl group analogous to the in vivo phosphorylated form of CheY. The mutation and alkylation was performed on D54C/C81S CheY. During the course of studying this protein a growth was also preformed using Escherichia coli to over-express CheY. The new alkylated analog of in vivo CheY-phosphate (P-CheY) was subject to optimized crystallization following its production, purification, and identification (using reversed phase HPLC). There were two favorable crystallization conditions resulting in diffraction sized crystals to date. The first was 24 % PEG 6000, 20 mM MgCl2, 100 mM HEPES (pH 7.0), 150 mM (NH4)2SO4, and an appropriate amount of Milli-Q water to give a total well volume of 500 micro-liters, which yielded a crystal diffracting to 1.2 Ã…. The second condition was 26 % PEG 3400, 0.2 M (NH4)2SO4, 15 mM MgCl2, 100 mM HEPES (pH 7.0), and an appropriate amount of Milli-Q water to give a total well volume of 500 micro-liters, which yielded a crystal diffracting to 1.5 Ã…. The purpose of this study was to optimize crystallization conditions for P-CheY based on past experimental results using this protein. The growth and diffraction of these crystals will be used to determine the structure of this analog. Using this data, experimentation involving co-crystallization of this protein with other phosphatases will lead to the long term goal of understanding how CheY-phosphate brings about a change in flagellum rotation upon binding the flagellar motor.