P U R I F I C A T I O N A N D F U N C T I O N A L CHARACTERIZATION OF A CYS-LESS VERSION OF Bacillus subtilis THERMOSENSOR

SAITA, E; MARTIN, M,; DE MENDOZA, D.

Potrero de los Funes, San Luis

Congreso; 47 Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2011

Histidine kinases (HKs) play a major role in signal transduction in prokaryotes for the cellular adaptation to environmental conditions and stresses. The HK DesK of Bacillus subtilis is a member of the two component system DesK/DesR responsible for Bacillus adaptation to cold shock. Its sensor region is confined to the five transmembrane segments (TMS) whose unknown  conformational rearrangements are essential for sensing and transducing the cold signal to the cytoplasmic catalytic domain. Site-directed spin labeling (SDSL) and Electronic ParamagneticResonance (EPR) have proven to be useful for elucidating structures of a great variety of integral membrane proteins. Since these techniques require having only one cysteine (Cys) in the protein at the site of study and DesK catalytic domain has two Cys residues, which seemed to be essential for the kinase activity, we first generated a Cys-less variant of DesK by overlap PCR. Then we found conditions in which DesKCys-less can be expressed and purified in a soluble form at high yields from E. coli cells. Finally, the biochemical characterization showed that DesKCys-less enzyme has less autokinase and phosphotransferase activities than DesK wild type, but it still kept cold shock regulation. These results will allow us to initiate the determination of DesK TMS folds and conformational dynamics using spin-labeling EPR spectroscopy.