In vitro reconstitution of the chimerical termosensor MS-DesK.

INDA, MARÍA EUGENIA; CYBULSKI, LARISA E.; DE MENDOZA, DIEGO

Tucumán, Argentina

Congreso; XLV Reunión Anual de la Sociedad Argentina de Bioquímica y Biología Molecular (SAIB); 2009

SAIB

Membrane proteins play a major role in communicating with the environment by transmitting signals across membranes. While most sensors detect the presence of a chemical signal, our thermosensor DesK of Bacillus subtilis detects physical changes in the membrane in response to temperature shifts. In an attempt to understand the novel mechanism that triggers the activation of DesK, we worked with the purposely minimalized sensor, MS-DesK. The MS captures into one single chimerical transmembrane segment (TMS) the essence of the whole sensor having 5 TMS. Peculiarly, a group of hydrophilic aminoacids (Q9, K10, N12) are located in the N-terminal region, near to the interfase lipid-water. Site directed mutagenesis revealed that these aminoacids are needed for the detection of a temperature downshift. How could these aminoacids determine the kinase to phosphatase activity of DesK? We reconstituted the MS in unilamellar liposomes and studied the effect of temperature on MS activity. We found that a temperature downshift stimulates MS kinase activity. We also analyzed the role of unusual aminoacids comparing the activities of MS-mutants in vitro. This simplified system developed now opens up the way to unravel in molecular detail the mechanism of temperature sensing.