CONICET | Buscador de Institutos y Recursos Humanos

¿How does thermosensors detect cold? Characterization of the chimericalthermosensor DesK-SM integrated in liposomes.Ma. Eugenia Inda, Larisa E. Cybulski, Diego de Mendoza.De p ar tamento d e Mi c ro biologí a, Uni v e r s id ad Na c ion al d e Ro s ar io , Su ip ac ha 5 31, 2000-Ro s ar i o, Ar g en t i na .in da@i br . go v .a rThermosensors are ubiquitous integral membrane proteins found in all kinds of life. They are involved in manyphysiological roles, including membrane remodeling, chemotaxis, touch and pain, but how can transmembrane domainssense cold stimulus? It remains largely unknown. Bacillus subtilis thermosensor DesK detects physical changes in themembrane in response to temperature shifts. To simplify the system, we worked with the purposely minimalized sensor,MS-DesK. The MS captures into one single chimerical transmembrane segment (TMS) the essence of the whole sensorhaving 5 TMS. Unusually, the N-terminus of MS has a cluster of hydrophilic aminoacids (Q9, K10, N12) near the lipidwaterinterfase. Site directed mutagenesis revealed that these aminoacids are needed for the detection of a temperaturedownshift. This suggests a hydration/stabilization switch implicated in temperature sensing so that an unstable state withdehydration of the polar groups in an expanding membrane is associated with the kinase activity, while stabilizationthrough hydration in a narrowing membrane would promote phosphatase activity. To test this idea, we reconstituted theMS in unilamellar liposomes and studied the effect of temperature on MS activities. We found that a temperaturedownshift stimulates MS kinase activity, while transition to a gel phase does not. We also studied in vitro the behavior ofa MS-mutant with an extra lysine in the hydrophilic cluster. This modification enhances the kinase activity at 25°C and42°C suggesting that the DesK signaling range can be genetically modified.