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.

Overexpression of Different Types of Microbial Rhodopsins with a Highly Expressible Bacteriorhodopsin from Haloarcula marismortui as a Single Protein in E. coli

“…The same phenomenon was observed in NpHR (Fig. 4c , middle), which was reported to undergo a proton circulation on the cytoplasmic side 27 . The same measurement was conducted on the HEBR-NpHR fusion protein (Fig.…”

“…The functionality of HEBR-NpHR was probed with the whole cell E. coli pH and protein-based photocurrent assays that we previously adopted and developed 27 . After expressing HEBR-NpHR in E. coli , the cells were centrifuged and resuspended in a non-buffer solution.…”

“…A slight modification of the electrochemical cell designed by Chu et al . 33 was used for light-driven photocurrent measurements, and a detailed description can be found in our previous study 27 . Briefly, a 0.5-W 532-nm continuous laser was used to stimulate M-Rho located in the photochemical cell, which was composed in the following order: ITO-coated glass slide, sample chamber, dialysis membrane, blank solution chamber, and then another ITO-coated slide.…”

Natronomonas salsuginis sp. nov., a New Inhabitant of a Marine Solar Saltern

“…Their rhodopsins and their applications have been deeply studied. Besides, several studies have focused on the biotechnological potential of its enzymes [7][8][9][10][11][12][13][14][15]. On the other hand, several culture-independent studies have also shown the presence of members of the genus Natronomonas in different environments from widely separated sites, such as hypersaline and alkaline lakes, solar salterns, saline-alkaline soils, and sediments or oil fields [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31], supporting the widespread distribution of species of this genus.…”

The Blue-Green Sensory Rhodopsin SRM from Haloarcula marismortui Attenuates Both Phototactic Responses Mediated by Sensory Rhodopsin I and II in Halobacterium salinarum

“…Second, halorhodopsin, a light-driven inward chloride pump activated by 580 nm light, is to maintain cellular osmotic pressure under extreme salinity 6 . In addition, we recently reported that halorhodopsin pumps chloride through a proton partnership 7 and thus, its physiological role requires more investigation. The third and fourth types of rhodopsins are the red light-sensing (585 nm) sensory rhodopsin I (SRI) and the blue light-sensing (485 nm) sensory rhodopsin II (SRII), which mediate photo-attractant and photorepellent responses, respectively 8–10 .…”