Dataset Information

A Unique Light-Driven Proton Transportation Signal in Halorhodopsin from Natronomonas pharaonis.

ABSTRACT: Halorhodopsin (HR) is a seven-transmembrane retinylidene protein from haloarchaea that is commonly known to function as a light-driven inward chloride pump. However, previous studies have indicated that despite the general characteristics that most HRs share, HRs from distinct species differ in many aspects. We present indium-tin-oxide-based photocurrent measurements that reveal a light-induced signal generated by proton release that is observed solely in NpHR via purified protein-based assays, demonstrating that indeed HRs are not all identical. We conducted mutagenesis studies on several conserved residues that are considered critical for chloride stability among HRs. Intriguingly, the photocurrent signals were eliminated after specific point mutations. We propose an NpHR light-driven, cytoplasmic-side proton circulation model to explain the unique light-induced photocurrent recorded in NpHR. Notably, the photocurrent and various photocycle intermediates were recorded simultaneously. This approach provides a high-resolution method for further investigations of the proton-assisted chloride translocation mechanism.

SUBMITTER: Chen XR

PROVIDER: S-EPMC5192691 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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A Unique Light-Driven Proton Transportation Signal in Halorhodopsin from Natronomonas pharaonis.

Chen Xiao-Ru XR Huang Yuan-Chi YC Yi Hsiu-Ping HP Yang Chii-Shen CS

Biophysical journal 20161201 12

Halorhodopsin (HR) is a seven-transmembrane retinylidene protein from haloarchaea that is commonly known to function as a light-driven inward chloride pump. However, previous studies have indicated that despite the general characteristics that most HRs share, HRs from distinct species differ in many aspects. We present indium-tin-oxide-based photocurrent measurements that reveal a light-induced signal generated by proton release that is observed solely in NpHR via purified protein-based assays, ...[more]

PMID: 28002736

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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

A Unique Light-Driven Proton Transportation Signal in Halorhodopsin from Natronomonas pharaonis.

Publications

A Unique Light-Driven Proton Transportation Signal in Halorhodopsin from Natronomonas pharaonis.

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