Structural Changes of pharaonis Phoborhodopsin upon Photoisomerization of the Retinal Chromophore:  Infrared Spectral Comparison with Bacteriorhodopsin

Abstract

Archaeal rhodopsins possess a retinal molecule as their chromophores, and their light energy and light signal conversions are triggered by all-trans to 13-cis isomerization of the retinal chromophore. Relaxation through structural changes of the protein then leads to functional processes, proton pump in bacteriorhodopsin and transducer activation in sensory rhodopsins. In the present paper, low-temperature Fourier transform infrared spectroscopy is applied to phoborhodopsin from Natronobacterium pharaonis (ppR), a photoreceptor for the negative phototaxis of the bacteria, and infrared spectral changes before and after photoisomerization are compared with those of bacteriorhodopsin (BR) at 77 K. Spectral comparison of the C--C stretching vibrations of the retinal chromophore shows that chromophore conformation of the polyene chain is similar between ppR and BR. This fact implies that the unique chromophore-protein interaction in ppR, such as the blue-shifted absorption spectrum with vibrational fine structure, originates from both ends, the beta-ionone ring and the Schiff base regions. In fact, less planer ring structure and stronger hydrogen bond of the Schiff base were suggested for ppR. Similar frequency changes upon photoisomerization are observed for the C==N stretch of the retinal Schiff base and the stretch of the neighboring threonine side chain (Thr79 in ppR and Thr89 in BR), suggesting that photoisomerization in ppR is driven by the motion of the Schiff base like BR. Nevertheless, the structure of the K state after photoisomerization is different between ppR and BR. In BR, chromophore distortion is localized in the Schiff base region, as shown in its hydrogen out-of-plane vibrations. In contrast, more extended structural changes take place in ppR in view of chromophore distortion and protein structural changes. Such structure of the K intermediate of ppR is probably correlated with its high thermal stability. In fact, almost identical infrared spectra are obtained between 77 and 170 K in ppR. Unique chromophore-protein interaction and photoisomerization processes in ppR are discussed on the basis of the present infrared spectral comparison with BR.

Steric Constraint in the Primary Photoproduct of an Archaeal Rhodopsin from Regiospecific Perturbation of C−D Stretching Vibration of the Retinyl Chromophore

“…FTIR spectroscopy was performed as described previously (19,20). Ninety microliters of the SRII sample was dried on a BaF 2 window with a diameter of 18 mm.…”

“…Ninety microliters of the SRII sample was dried on a BaF 2 window with a diameter of 18 mm. The dry film was then rehydrated by putting an ∼1 µL drop of water beside the film and sealing with another window and a silicon rubber O-ring (19,20). After hydration, the sample was placed in a cell, which was mounted in an Oxford DN-1704 cryostat placed in the Bio-Rad FTS-40 spectrometer.…”

Functional Importance of the Interhelical Hydrogen Bond between Thr204 and Tyr174 of Sensory Rhodopsin II and Its Alteration during the Signaling Process

“…Binding Analysis and FTIR Measurements-SRII and HtrII-(1-159) proteins were prepared as described previously (26,27). A truncated HtrII protein expressed from position 1 to position 159, which has been shown to exhibit the same interaction with SRII as the full-length protein (28,29), was used for measurements other than phototaxis behavior and flash photolysis.…”

Dynamic structure of pharaonis phoborhodopsin (sensory rhodopsin II) and complex with a cognate truncated transducer as revealed by site‐directed 13C solid‐state NMR

“…In addition, transient movement of helix F in ppR and receptort ransducer signal transfer were analyzed by means of electron paramagnetic resonance study on spin-labeled ppR and Htr II and their complex [10,11]. In contrast, we have demonstrated that a site-directed solid-state 13 C nuclear magnetic resonance (NMR) approach provides an alternative means to delineate conformation and dynamics of 13 C-labeled bR as a typical membrane protein at ambient temperature, once resolved 13 C signals, if any, could be assigned to certain residues of interest utilizing site-directed mutants [12^14] and conformation-dependent displacement of 13 C chemical shifts [15,16]. This approach turned out to be especially useful as a complementary means to di¡raction studies in which structural data of surfaces such as C-or N-terminals as well as loop regions are in many instances lost owing to a disordered or motionally averaged state [12^14].…”

“…[3-13 C]Ala, [1-13 C]Val-labeled ppR with a histidine-tag at the C-terminus expressed in Escherichia coli BL21 (DE3) strain was grown in the M9 medium to which 13 C-labeled L-Ala and L-Val were added and solubilized with 1.5% n-dodecyl L-D-maltoside (DM), followed by pu-ri¢cation with Ni-NTA resin (Qiagen) as described previously [15]. The puri¢ed protein was mixed with lipid ¢lm of egg PC (1:50 mol ratio) in which chloroform solvent was evaporated and DM as detergent was removed with Bio-Beads (SM2; Biorad) to yield ppR incorporated into egg PC bilayer.…”