Abstract

The two-component system DesK/DesR in *Bacillus subtilis* is a critical regulator of membrane fluidity, sensing changes in the lipid bilayer's physical state and initiating the cold-adaptation response. DesK, a sensor histidine kinase, is known to be allosterically regulated by its bound adenosine phosphate state, which influences its oligomerization and kinase/phosphatase activity. This study investigates the impact of recombinant expression temperature on the adenosine phosphate (AP) bound state of purified BsDesK, a factor crucial for obtaining a functionally homogeneous protein preparation for structural and biophysical analysis. Recombinant BsDesK was expressed at standard (37°C) and reduced (20°C) temperatures, and the resulting protein preparations were analyzed using size-exclusion chromatography and spectroscopic methods. Our findings demonstrate that lowering the expression temperature to 20°C significantly increases the proportion of the ATP-bound, oligomeric form of BsDesK, suggesting a direct link between the cellular environment during expression and the protein's functional conformation. This temperature-dependent shift in the AP-bound state provides a novel and effective strategy for optimizing the preparation of active membrane-associated sensor kinases, which is essential for advancing our understanding of two-component signaling mechanisms.