Light-Induced Switching of HAMP Domain Conformation and Dynamics Revealed by Time-Resolved EPR Spectroscopy

In microbial photo- and chemotaxis a two-component signaling cascade mediates a regulated response of the flagellar motor to environmental conditions. Upon activation, photo- and chemoreceptors transfer a signal across the plasma membrane to activate the histidine kinase CheA. Successive regulation of the CheY-phosphorylation level controls the flagellar motor.In Natronomonas pharaonis a sensory rhodopsin II - transducer complex (SRII/HtrII) mediates negative phototaxis. As the initial signal, a light-induced outward movement of receptor helix F leads to a conformational change of transducer helix TM2, which in turn propagates the signal to the adjacent HAMP domain.For the HAMP domain, a widely abundant signaling module, several mechanisms were suggested, all comprising two distinct conformational states which we previously observed by two-component cw-EPR spectra at ambient temperatures.Here, we trace the conformational signal and it's propagation throughout the elongated transducer.(1) We applied cw- and pulse-EPR spectroscopy in conjunction with nitroxide spin labeling. We follow transient changes by time-resolved cw-EPR spectroscopy and compare the resulting spectral changes to simulated EPR difference spectra revealing a shift in the thermodynamic equilibrium between the two states. Structure-based calculations of the expected spectral differences shows agreement with a shift towards a more compact state of the HAMP domain.To extend the current signaling models to the whole complex, a trimer of NpSRII/NpHtrII dimers, we carried out molecular dynamics simulations and observed differences between the deactivated and activated complex, ultimately leading to a signaling model that can now be tested experimentally.[1] Klose, D. et al., FEBS Lett. (2014) http://dx.doi.org/10.1016/j.febslet.2014.09.012 (in press)