Synthesis and characterization of phosphono-CheY from Thermotoga maritima

The chemical and molecular mechanisms of bacterial chemotaxis have been extensively characterized through structural studies of mutant chemotaxis proteins. CheY~P (the active, signaling form of CheY) binds the switch protein FliM at the intracellular base of the flagellar motor, switching the direction of flagellar rotation from clockwise to counterclockwise and punctuating periods of smooth-swimming with tumbling and random reorientation. The conformational changes accompanying phosphorylation and activation of the response regulator CheY have been identified; however, transfer of the “active” signal from CheY~P to the flagellar switch complex is unknown. Crystallization of FliM alone and in complex with CheY~P would allow detailed analysis of the interactions between CheY~P and FliM that force a change in the directional rotation of the flagella. These structures are difficult to crystallize since FliM is a membrane-bound protein and is not soluble in the conditions necessary for crystallization. The a-proteobacteria, Bacillus subtilis and Thermotoga maritima contain protein phosphatases that share homology with the central domain of FliM. If these phosphatases bind CheY~P in a similar manner as FliM, a crystal structure of CheY~P in complex with one of these phosphatases may shed light on the interactions that occur at the base of the flagellar motor. Since CheY~P has a short half life, an analogue known as phosphono-CheY was synthesized and purified under various conditions. Thermotoga maritima D54C/C81S CheY was purified from the soluble fraction of a cell lysate using immobilized metal affinity chromatography (Ni2+-NTA) and size-exclusion chromatography. Purified CheY was reacted with phosphonomethyltriflate under alkaline conditions in the presence of a divalent (or trivalent) metal yielding roughly 50% phosphono-CheY by RP-HPLC and reaction with Ellman’s reagent. Phosphono-CheY was separated from un-reacted CheY by first labeling the unmodified protein with biotin and then isolating the two species by avidin affinity chromatography or by hydrophobic interaction chromatography. The presence of phosphono-CheY was confirmed by electrospray ionization mass spectrometry of the intact protein. Mass spectrometry also suggested the presence of multiply-phosphonomethylated protein and oxidized protein within a phosphonomethylation reaction mixture and experiments to disprove this hypothesis are on-going.