Steric Constraint in the Primary Photoproduct of Sensory Rhodopsin II Is a Prerequisite for Light-Signal Transfer to HtrII

Abstract

Sensory rhodopsin II (SRII, also called pharaonis phoborhodopsin, ppR) is responsible for negative phototaxis in Natronomonas pharaonis. Photoisomerization of the retinal chromophore from all-trans to 13-cis initiates conformational changes in the protein, leading to activation of the cognate transducer protein (HtrII). We previously observed enhancement of the C 14 -D stretching vibration of the retinal chromophore at 2244 cm −1 upon formation of the K state and interpreted that a steric constraint occurs at the C 14 D group in SRII K . Here, we identify the counterpart of the C 14 D group as Thr204, because the C 14 -D stretching signal disappeared in T204A, T204S, and T204C mutants as well as a C 14 -HOOP (hydrogen out-of-plane) vibration at 864 cm −1 . Although the K state of the wild-type bacteriorhodopsin (BR), a light-driven proton pump, possesses neither 2244 nor 864 cm −1 bands, both signals appeared for the K state of a triple mutant of BR that functions as a light sensor (P200T/V210Y/A215T). We found a positive correlation between these vibrational amplitudes of the C 14 atom at 77 K and the physiological phototaxis response. These observations strongly suggest that the steric constraint between the C 14 group of retinal and Thr204 of the protein is a prerequisite for light-signal transduction by SRII.Microbial rhodopsins convert light into chemiosmotic energy or cellular signals (1, 2). The primary reaction is all-trans to 13-cis photoisomerization of the retinal chromophore, and light energy is stored through altered chromophore-protein interaction (2, 3). Such energy is subsequently consumed by changing the protein structures, and ion pumping or transducer activation is achieved in light-energy or signal conversions, respectively. It is well-known that retinal isomerization in the protein is a selective and efficient reaction along the

Active State of Sensory Rhodopsin II: Structural Determinants for Signal Transfer and Proton Pumping

“…Unfortunately, due to the resolution of diffraction data, we are not able to observe neither the minute changes in the structure nor movements of the water molecules in the cytoplasmic channel. Nonetheless we are able to make a conclusion, that there are no changes in the uncomplexed NpSRII active state in the relative positions of the residues T204 and Y174 (Figure 3), which are proposed to be important for the signal generation in NpSRII/NpHtrII complex 42,43 . Probably, this reflects the spectroscopically observed absence of the alteration in T204-Y174 bonding in uncomplexed NpSRII 44,45 .…”

Protein‐Controlled Ultrafast Photoisomerization in Rhodopsin and Bacteriorhodopsin

“…Although the wild -type BR shows no enhanced C14 -D stretch, a similar band appeared in a triple mutant (P200T/V210Y/A215T) possessing Thr215. 98 The mutant BR has a sensor function, 99 and a positive correlation was observed between the C14 -D stretching signal and phototaxis. 98 takes place at the center of the chromophore, whereas no changes occur at the Schiff base.…”

“…97 By use of a mutant, the counterpart of the C14 -D group was determined to be Thr204. 98 Although the K state of the wild -type BR does not possess the 2244 -cm − 1 band, the band newly appeared for the K state of a triple mutant of BR that functions as a light sensor (P200T/V210Y/A215T). 99 We found a positive correlation between the vibrational amplitude of the C14 atom at 77 K and the physiological phototaxis response.…”

“…These observations strongly suggest that the steric constraint between the C14 group of retinal and Thr204 of the protein (Thr215 for a triple mutant of BR) is prerequisite for light -signal transduction by SRII. 98 Thus, photoisomerization -induced steric constraint between the C14 -H of the retinal chromophore and Thr204 in SRII is one such mechanism. It is likely that a water -containing hydrogen -bonding network is important for the energy storage in proton pumps like BR, while steric constraint is important for the energy storage in sensor functions as seen for visual Rh, SRII, and a BR mutant as a light sensor.…”

“…In visual Rhs, conformational distortion 7,8,10,11,12,14,15), only the C14 -D stretching vibration at 2244 cm − 1 is signifi cantly enhanced upon formation of the K state of SRII, 97 where the counterpart in the protein side is Thr204. 98 The corresponding amino acid in BR is Ala215. Although the wild -type BR shows no enhanced C14 -D stretch, a similar band appeared in a triple mutant (P200T/V210Y/A215T) possessing Thr215.…”