FTIR Study of the L Intermediate of Anabaena Sensory Rhodopsin: Structural Changes in the Cytoplasmic Region

Abstract

Anabaena sensory rhodopsin (ASR) is an archaeal-type rhodopsin found in eubacteria. The gene encoding ASR forms a single operon with ASRT (ASR transducer) that is a 14 kDa soluble protein, suggesting that ASR functions as a photochromic sensor by activating the soluble transducer. One of the characteristics of ASR is that the formation of the M intermediate accompanies a proton transfer from the Schiff base to Asp217 in the cytoplasmic side [Shi, L., Yoon, S. R., Bezerra, A. G., Jr., Jung, K. H., and Brown, L. S. (2006) J. Mol. Biol. 358, 686-700], in remarkable contrast to other archaeal-type rhodopsins such as a light-driven proton-pump, bacteriorhodopsin (BR). In this study, we applied low-temperature Fourier transform infrared (FTIR) spectroscopy to the all- trans form of ASR at 170 K, and compared the structural changes in the L intermediate with those of BR. The ASR L minus ASR difference spectra were essentially similar to those for BR, suggesting common structures for the L state in ASR and BR. On the other hand, unique CO stretching bands of a protonated carboxylic acid were observed at 1722 (+) and 1703 (-) cm (-1) at pH 5 and 7, and assigned to Glu36 by use of mutants. Glu36 is located at the cytoplasmic side, and the distance from the Schiff base is about 20 A. This result shows the structural changes at the cytoplasmic surface in ASR L. pH-dependent frequency change was also observed for a water stretching vibration, suggesting that the water molecule is involved in a hydrogen-bonding network with Glu36 and Asp217. Unique hydrogen-bonding network in the cytoplasmic domain of ASR will be discussed.

Structure of an Inward Proton-Transporting Anabaena Sensory Rhodopsin Mutant: Mechanistic Insights

“…Although both the crystal structures of D217E and WT ASR suggest that Glu36 is protonated due its hydrogen bonding with Gln93, there are inherent limitations in discussing water-exposed hydrogen bonds based on crystallographic structures solved at cryogenic temperatures. Previous FTIR studies with WT ASR not only showed an alteration of hydrogen bonding of a water but also of Glu36, 20 Å from the Schiff base, as early as during the formation of the L state at 170K (42). This work also demonstrated that Glu36 WT displays an elevated pK a (between 6 and 7).…”

“…It has been shown that upon photon absorption, the Schiff base proton is transferred to Asp217 in the cytoplasmic half (39), 15 Å from the retinal. However, because there is no significant net proton transfer through the protein, as determined by the monitoring of light-induced pH changes in nonbuffered solutions containing ASR-imbedded spheroplasts (40), it has been postulated that the Schiff base is reprotonated from the same residue (41,42). As in most microbial rhodopsins, with the exception of proteorhodopsin (43), there is a tightly coordinated water molecule (Wat402) in the extracellular domain that is strongly hydrogen-bonded to the retinal Schiff base in the protonated state, while it is only weakly hydrogen-bonded to its counterion Asp75 (Asp85 in BR), which does not participate in reprotonation (41).…”

Photo-induced Regulation of the Chromatic Adaptive Gene Expression by Anabaena Sensory Rhodopsin

“…The transmembrane helices (pink), the retinal (yellow), and the extended C-terminal region are shown according to the crystal structure of the truncated protein (19). The structure of the 35 cytoplasmic residues beyond residue 226 is unknown and has been predicted to be an ␣-helical extension.…”

“…ASR is encoded in an operon, along with a second gene that encodes a small soluble cytoplasmic protein, tentatively named the ASR transducer (ASRT) (10), which is different from the members of the membrane-embedded taxis transducer family proteins, haloarchaeal phototaxis transducers I for SRI and haloarchaeal phototaxis transducers II for SRII (11,12). The photochemical properties of ASR and the interaction between ASR and ASRT have already been studied (9,10,(13)(14)(15)(16)(17)(18)(19), but the physiological function of both ASR and ASRT in Anabaena is still poorly understood, although isothermal titration calorim-etry measurements revealed that ASRT binds to ASR with a dissociation constant of 8 M (10). The crystallographic analysis of ASR has revealed its photochromic nature and the existence of a water molecule network in its cytoplasmic half, where it possibly interacts with ASRT (20).…”

A role of Anabaena sensory rhodopsin transducer (ASRT) in photosensory transduction

“…The retinal Schiff base deprotonation, which occurs in the M intermediate phase of the photocycle of all-trans-ASR, results in the protonation of Asp217 on the cytoplasmic side of the protein. The water molecule is involved in a hydrogen-bonding network with Glu36 and Asp217 on the cytoplasmic side of the protein (Shi et al, 2006;Sineshchekov et al, 2006;Kawanabe et al, 2008). The cytoplasmic shuttling of protons in the photocycle of the all-trans-ASR and the ensuing conformational changes might act as a trigger to activate the transducer.…”