The photoactivation mechanism of the sensory rhodopsin II (SRII)-HtrII receptor-transducer complex of Natronomonas pharaonis was investigated by time-resolved Fourier transform infrared difference spectroscopy to identify structural changes associated with early events in the signal relay mechanism from the receptor to the transducer. Several prominent bands in the wild-type SRII-HtrII spectra are affected by amino acid substitutions at the receptor
Sensory rhodopsin II (SRII)1 functions as a phototaxis receptor for blue light avoidance in several halophilic archaea (1-4). This protein belongs to a growing family of microbial rhodopsins, seven-helix retinylidene membrane proteins now found in all domains of life (5-8). The SRII receptor forms a tight intermolecular complex with its cognate transducer protein HtrII in the cell membrane. The transducer, like its homologous methyl-accepting chemotaxis transducers, possesses two transmembrane helices and a large cytoplasmic domain that binds at its distal end a His-kinase that phosphorylates a flagellar motor switch regulator (1, 9).Interactions of HtrII with the SRII receptor are localized to the transducer transmembrane and membrane-proximal domains (10, 11). A crystal structure of SRII bound to an Nterminal HtrII fragment containing the transmembrane helices (TM1 and TM2) shows tight van der Waals interaction and three hydrogen bonds between TM2 and SRII helices F and G (12). An atomic structure of the membrane-proximal domain of the transducer is not available; however, fluorescent probe accessibility and Förster resonance energy transfer measurements show interaction of this domain with the cytoplasmic E-F loop of the receptor (13).The signal relay mechanism from SRII to HtrII in the complex has become a focus of interest in the past several years, in part because of its importance to the general understanding of interaction between integral membrane proteins. In accord with the unified model for transport and signaling by microbial rhodopsins (14, 15), the key event in the transducer activation is an outward tilt of the receptor helix F during the lifetime of its M intermediate, which has been directly detected by EPR of paramagnetic probes in the free receptor (16) and in its complex with transducer (17). In a response to the helix F movements, the TM2 of HtrII is displaced from its initial position (17). Additional structural changes were observed in the cytoplasmic membrane proximal region by fluorescent probe accessibility measurements (13).Fourier transform infrared (FTIR) difference spectroscopy has been used extensively in the past to elucidate structural changes in the photocycles of several microbial rhodopsins (18 -27). This technique measures changes in the infrared absorption of protein groups and enables studies of light-induced conformational changes without the necessity of introducing potentially structure-perturbing probes. Because of its sensitivity to small changes in hydrogen bonding, it is especially well suited for the study of interacti...