Effect of a bound anion on the structure and dynamics of halorhodopsin from Natronomonas pharaonis

Abstract

Active ion transport across membranes is vital to maintaining the electrochemical gradients of ions in cells and is mediated by transmembrane proteins. Halorhodopsin (HR) functions as a light-driven inward pump for chloride ions. The protein contains all-trans-retinal bound to a specific lysine residue through a protonated Schiff base. Interaction between the bound chloride ion and the protonated Schiff base is crucial for ion transport because chloride ion movement is driven by the flipping of the protonated Schiff base upon photoisomerization. However, it remains unknown how this interaction evolves in the HR photocycle. Here, we addressed the effect of the bound anion on the structure and dynamics of HR from Natronomonas pharaonis in the early stage of the photocycle. Comparison of the chloride-bound, formate-bound, and anion-depleted forms provided insights into the interaction between the bound anion and the chromophore/protein moiety. In the unphotolyzed state, the bound anion affects the π-conjugation of the polyene chain and the hydrogen bond of the protonated Schiff base of the retinal chromophore. Picosecond time scale measurements showed that the band intensities of the W16 and W18 modes of the tryptophan residues decreased instantaneously upon photoexcitation of the formate-bound form. In contrast, these intensity decreases were delayed for the chloride-bound and anion-depleted forms. These observations suggest the stronger interactions of the bound formate ion with the retinal chromophore and the chromophore pocket. On the nanosecond to microsecond timescales, we found that the interaction between the protonated Schiff base and the bound ion is broken upon formation of the K intermediate and is recovered following translocation of the bound anion toward the protonated Schiff base in the L intermediate. Our results demonstrate that the hydrogen-bonding ability of the bound anion plays an essential role in the ion transport of light-driven anion pumps.

Unique Vibrational Characteristics and Structures of the Photoexcited Retinal Chromophore in Ion-Pumping Rhodopsins

“…The CN/CC stretching band was observed at 1597 cm –1 for the S 1 -state chromophore in Np HR. This frequency is in sharp contrast to the CN stretching frequencies for the unphotolyzed state and the ground-state intermediates of microbial rhodopsins, which are reported to range from 1620 to 1660 cm –1 . − ,,− ,,,,,− Moreover, the CC stretching frequencies at 1400–1500 cm –1 for the S 1 -state chromophore are much lower than those for the unphotolyzed state and the ground-state intermediates. Conversely, the C–C stretching frequency at 1251 cm –1 for the S 1 -state chromophore was substantially higher than those for the unphotolyzed state and the ground-state intermediates.…”

“…Based on their fast decay rates, it is reasonable to attribute the decaying bands in the present time window to the S 1 state. The spectral patterns of the S 1 bands were completely different from those of the ground-state chromophore, ,− indicating the unique structure of the S 1 -state chromophore. The RR spectrum observed for the S 1 -state chromophore in Np HR was similar to that reported for Hs BR measured within the instrument response time using probe pulses under the resonant conditions with the excited-state absorptions.…”

“…The deuteration downshift of 13 cm –1 and the highest relative intensity of the 1597 cm –1 band for the S 1 -state chromophore in Np HR suggest that this mode contains CN stretching and CC stretching characteristics. No RR band showing both a deuteration downshift larger than 10 cm –1 and highest relative intensity was observed in RR spectra of the unphotolyzed state and of ground-state intermediates for microbial rhodopsins. − ,,− ,,,,,− Thus, the S 1 -state chromophore in Np HR exhibits unprecedentedly strong vibrational mixing of the CN and CC stretching modes. In contrast, no clear mixing of a CC stretching characteristic to the CN stretching mode is seen in the QM/MM data (Figure ).…”

“…We analyzed the Raman bands of the S 1 -state chromophore at 1597, 1251, and 873 cm –1 , corresponding to the CN/CC stretching, C–C stretching, and HOOP modes, respectively. The intensities and vibrational frequencies of these bands differ from those of the ground-state species, including the unphotolyzed state and ground-state intermediates. − ,,− ,,,, Those bands were successfully assigned based on a combination of observations of the deuteration shifts and QM/MM calculation results. Although a quantitatively accurate vibrational frequency calculation that includes anharmonic vibrational mode couplings is very computationally demanding and is formidable for many vibrational modes in both the S 1 excited state and in the unphotolyzed state, we succeeded in qualitative assignment of the vibrational modes using conventional normal-mode analysis by focusing on the deuteration effects and combining with experimental observations.…”

Concerted Motions and Molecular Function: What Physical Chemistry We Can Learn from Light-Driven Ion-Pumping Rhodopsins

“…Steric interactions between methyl groups and the amino acid side chains have been detected both by spectroscopy and crystallography. For BR, the interaction between a methyl group and the side chain of Trp182 was detected for the L intermediate in low-temperature FTIR spectra. , Similar steric collisions were observed for the M intermediate using time-resolved X-ray crystallography. , Changes in ultraviolet resonance Raman spectra on the picosecond time scale were observed for the tryptophan and tyrosine residues contacting the polyene chain. − These spectral changes were found for the five microbial rhodopsins studied, suggesting that structural changes around the tryptophan residues are induced by atomic contacts between the retinal chromophore and the surrounding amino acid residues.…”