Dataset Information

Detection of a protein-bound water vibration of halorhodopsin in aqueous solution.

ABSTRACT: Protein-bound water molecules play crucial roles in their structure and function, but their detection is an experimental challenge, particularly in aqueous solution at room temperature. By applying attenuated total reflection (ATR) Fourier-transform infrared (FTIR) spectroscopy to a light-driven Cl(-) pump pharaonis halorhodopsin (pHR), here we detected an O-H stretching vibration of protein-bound water molecules in the active center. The pHR(Cl(-)) minus pHR(Br(-)) ATR-FTIR spectra show random fluctuation at 3600-3000 cm(-1), frequency window of water vibration, which can be interpreted in terms of dynamical fluctuation of aqueous water at room temperature. On the other hand, we observed a reproducible spectral feature at 3617 (+)/3630 (-) cm(-1) in the pHR(Cl(-)) minus pHR(Br(-)) spectrum, which is absent in the pHR(Cl(-)) minus pHR(Cl(-)) and in the pHR(Br(-)) minus pHR(Br(-)) spectra. The water O-H stretching vibrations of pHR(Cl(-)) and pHR(Br(-)) at 3617 and 3630 cm(-1), respectively, are confirmed by light-induced difference FTIR spectra in isotope water (H2 (18)O) at 77 K. The observed water molecule presumably binds to the active center of pHR, and alter its hydrogen bond during the Cl(-) pumping photocycle.

SUBMITTER: Fukuda T

PROVIDER: S-EPMC4629683 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Detection of a protein-bound water vibration of halorhodopsin in aqueous solution.

Fukuda Tetsuya T Muroda Kosuke K Kandori Hideki H

Biophysics (Nagoya-shi, Japan) 20131221

Protein-bound water molecules play crucial roles in their structure and function, but their detection is an experimental challenge, particularly in aqueous solution at room temperature. By applying attenuated total reflection (ATR) Fourier-transform infrared (FTIR) spectroscopy to a light-driven Cl(-) pump pharaonis halorhodopsin (pHR), here we detected an O-H stretching vibration of protein-bound water molecules in the active center. The pHR(Cl(-)) minus pHR(Br(-)) ATR-FTIR spectra show random ...[more]

PMID: 27493555

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Project description: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 ?-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.

Dataset Information

Detection of a protein-bound water vibration of halorhodopsin in aqueous solution.

Publications

Detection of a protein-bound water vibration of halorhodopsin in aqueous solution.

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