In this work, we studied three proteins from three different organisms by EPR spectroscopy: NpHtrII is part of the phototaxis system found in halophilic archaea, ChR2 is a cation-selective channel isolated from a green alga, and HsaCCA is an enzyme involved in the protein biosynthesis of humans. The goal was to identify characteristic conformations and dynamics in each of the studied proteins that were linked to their specific functions. The scientific disciplines employed in this work include biochemistry (site-directed spin-labeling), bioinformatics (data analysis, molecular modeling), informatics (device control, software development), molecular biology (mutagenesis, transformation, heterologous protein expression, protein purification, protein characterization), and physics (EPR spectroscopy, optical spectroscopy, experimental assembly). The experimental results show a strong interdependence between protein structure, conformers, dynamics, and function. Hydrogen bonds, although being a transient electrostatic attraction between polar molecules, are the key molecular interactions required for the conservation of protein functionality: Hydrogen bond networks in NpSRII and ChR2 stabilize the helix bundles, and hydrogen bond networks in HsaCCA mediate interdomain flexibility. However, the resulting structural alterations observed in our proteins manifest on a much larger scale: We have detected changes in the protein backbone mobility of the HAMP2 domain in NpHtrII after signaling. We have discovered the TMH B movement in ChR2 accompanying channel opening. And we have documented a substrate-dependent motion of the head domain in HsaCCA during catalysis.

The signal transduction pathway of halophilic archaea remains a fascinating example of adaptation to extreme environments. Despite similarities with bacterial taxis systems, its structural and dynamics patterns during signal relay remain debatable. The currently investigated SRII/HtrII phototaxis system of Natronomonas pharaonis shows remarkable similarities with chemoreceptors in its membrane and HAMP domains functioning design. By combining site-directed spin labeling (SDSL) with electron paramagnetic resonance (EPR) spectroscopy we investigate the kinase control domain (i.e. signaling domain) of NpSRII/HtrII both in terms of dynamic and structural properties. Our data, as provided by continuous wave and pulse (DEER) EPR techniques, builds on current dynamics based signaling models for HAMP domains (such as the “frozen–dynamic” or two-state equilibrium models). We present an expanded mechanism for signal propagation throughout the signaling domain, where salt and temperature variations trigger subtle shifts in dynamics. Extreme dynamics motional ranges (compact or highly-dynamic) associate with a specific flagellar signaling state, here the kinase-off response, where a more moderate dynamics motion (dynamic) associates with the kinase-on response. Structurally, we reference our data on PML and ND reconstituted NpSRII/HtrII to the EcTsr crystal structure and the NpHtrII homology model. We show that, despite a difference in packing, NpHtrII oligomerizes in a similar manner as EcTsr, even in the absence of stabilizing structures such as the CheA/CheW baseplate. The presence of trimers-of-dimers but also dimers-of-dimers in membrane sheet samples exposes the high affinity with which NpHtrII signaling domains interact. We hope our structural and dynamics details will push further not just drug design but also environmental preservation efforts where taxis systems drive colonization and virulence of pathogens in plants, animals and humans alike.