Development and optimization of a purification protocol for active variants of DesK, the Bacillus subtilis thermosensor

SAITA, E; ALBANESI, D; MANSILLA, MC; DE MENDOZA, D

Congreso; XLVIII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2012

SAIB

Histidine kinases (HKs) play a major role in signal transduction in prokaryotes for cellular adaptation to environmental conditions and stresses. In Bacillus subtilis, the HK DesK constitutes, together with DesR and D5_Des, the Des pathway which is responsible for cell adaptation to cold shock. The sensor region of DesK is confined to the five transmembrane segments (TMS) and is essential for sensing and transducing the cold signal to the cytoplasmic catalytic domain through still unknown conformational rearrangements.Site-directed spin labeling (SDSL) and Electronic Paramagnetic Resonance (EPR) have proven to be useful for elucidating structures of a great variety of integral membrane proteins. These spectrometric techniques require proteins, containing a single cysteine residue, to be in a solution free of contaminating proteins implying that target proteins should be purified to homogeneity. To achieve this key issue we tested different expression systems, culture media, protease cleavage and chromatographic techniques.The molecular tools and purification steps applied in this work led us to develop a suitable protocol to purify the active variants of DesK to homogeneity. The purified variants of the thermosensor will make feasible to determine the dynamic of the lipid-induced TM helix rearrangements of DesK by means of spin-labeling EPR spectroscopy studies.