Environmental awareness is an essential attribute of all organisms. The homeoviscous adaptation system of Bacillus subtilis provides a powerful experimen tal model for the investigation of stimulus detection and signaling mechanisms at the molecular level. These bacteria sense the order of membrane lipids with the transmembrane (TM) protein DesK, which has an N-terminal sensor domain and an intracellular catalytic effector domain. DesK exhibits autokinase activity as well as phosphotransferase and phosphatase activities toward a cognate response regulator, DesR, that controls the expression of an enzyme that remodels membrane fluidity when the temperature drops below 30°C. Membrane fluidity signals are transmit ted from the DesK sensor domain to the effector domain via rotational movements of a connecting 2-helix coiled coil (2-HCC). Previous molecular dynamic simulations suggested important roles for TM prolines in transducing the initial signals of mem brane fluidity status to the 2-HCC. Here, we report that individual replacement of prolines in DesKs TM1 and TM5 helices by alanine (DesKPA) locked DesK in a phosphatase-ON state, abrogating membrane fluidity responses. An unbiased muta genic screen identified the L174P replacement in the internal side of the repeated heptad of the 2-HCC structure that alleviated the signaling defects of every trans membrane DesKPA substitution. Moreover, substitutions by proline in other internal positions of the 2-HCC reestablished the kinase-ON state of the DesKPA mutants. These results imply that TM prolines are essential for finely tuned signal generation by the N-terminal sensor helices, facilitating a conformational control by the meta stable 2-HCC domain of the DesK signaling state.