The recently identified Krokinobacter rhodopsin 2 (KR2) functions as a light-driven sodium ion pump. The structure of the retinal-binding pocket of KR2 offers important insights into the mechanisms of KR2, which has motif of Asn112, Asp116, and Gln123 (NDQ) that is common among sodium ion pump rhodopsins but is unique among other microbial rhodopsins. Here we present solid-state nuclear magnetic resonance (NMR) characterization of retinal and functionally important residues in the vicinity of retinal in the ground state. We assigned chemical shifts of retinal C14 and C20 atoms, and Tyr218Cζ, Lys255Cε, and the protonated Schiff base of KR2 in lipid environments at acidic and neutral pH. N NMR signals of the protonated Schiff base showed a twist around the N-Cε bond under neutral conditions, compared with other microbial rhodopsins. These data indicated that the location of the counterion Asp116 is one helical pitch toward the cytoplasmic side. In acidic environments, theN Schiff base signal was shifted to a lower field, indicating that protonation of Asp116 induces reorientation during interactions between the Schiff base and Asp116. In addition, the Tyr218 residue in the vicinity of retinal formed a weak hydrogen bond with Asp251, a temporary Na-binding site during the photocycle. These features may indicate unique mechanisms of sodium ion pumps.

Proton pumping ability of a light-driven Na+ pumping Krokinobacter rhodopsin 2 (KR2) is inhibited by H30A and lack of bound Na+ at extracellular site.

Photo-reaction pathways of a bacteriorhodopsin Y185F mutant were examined using in situ photo-irradiation solid-state NMR spectroscopy. (13)C CP MAS NMR spectra were recorded at -40 °C in the dark (D1), under irradiation with 520 nm light (L1), subsequently in the dark (D2), and again under irradiation with 520 nm light (L2). In the process from D1 to L1, the 13-cis, 15-syn (CS; bR548) state changed to a CS*- (13-cis, 15-syn) intermediate, which was highly stable at -40 °C, and the all-trans (AT; bR568) state transformed to an N-intermediate. Under the D2 conditions, the N-intermediate transformed to an O-intermediate, which was highly stable at -40 °C in the dark. During subsequent irradiation with 520 nm light (L2), the O-intermediate transformed to the N-intermediate through the AT state, whereas the CS*-intermediate did not change. The CS*-intermediate was converted to the AT state (or O-intermediate) after the temperature was increased to -20 °C. Upon subsequent increase of the temperature to 20 °C, the AT state (or O-intermediate) was converted to the CS state until reaching equilibrium. In this experiment, the chemical shift values of [20-(13)C, 14-(13)C]retinal provided the 13C[double bond, length as m-dash]C and 15C[double bond, length as m-dash]N configurations, respectively. From these data, the configurations of the AT and CS states and the CS*-, N-, and O-intermediates were determined to be (13-trans, 15-anti), (13-cis, 15-syn), (13-cis, 15-syn), (13-cis, 15-anti), and (13-trans, 15-anti), respectively. (13)C NMR signals of the CS*- and O-intermediates were observed for the first time for the Y185F bR mutant by in situ photo-irradiation solid-state NMR spectroscopy and the configuration of the CS*-intermediate was revealed to be significantly twisted from that of the CS state although both were assigned as (13-cis, 15-syn) configurations.

Photoirradiation solid-state NMR spectroscopy is a powerful means to study photoreceptor retinal-binding proteins by the detection of short-lived photointermediates to elucidate the photoreaction cycle and photoactivated structural changes. An in situ photoirradiation solid-state NMR apparatus has been developed for the irradiation of samples with extremely high efficiency to enable observation of photointermediates which are stationary trapped states. Such observation enables elucidation of the photoreaction processes of photoreceptor membrane proteins. Therefore, in situ photoirradiation is particularly useful study the photocycle of retinal-binding proteins such as sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) because functional photointermediates have relatively longer half-lives than other photointermediates. As a result, several photointermediates have been trapped as stationary state and their detailed structures and photoreaction cycles have been revealed using photoirradiation solid-state NMR spectroscopy at low temperature. Photoreaction intermediates of bacteriorhodopsin, which functions to provide light-driven proton pump activity, were difficult to trap because the half-lives of the photointermediates were shorter than those of sensory rhodopsin. Therefore, these photointermediates are trapped in a freeze-trapped state at a very low temperature and the NMR signals were observed using a combination of photoirradiation and dynamic nuclear polarization (DNP) experiments.