Characterization of two predicted bacteriorhodopsins in Haloquadratum walsbyi
Date Issued
2012
Date
2012
Author(s)
Liu, Hung-Yi
Abstract
Studies in microbial rhodopsins have been focused on those found in Halobacterium salinarum since 1973. In the past, the studies in halophilic archaea identified four kinds of retinal-binding proteins and they use light as energy source to mediate different physiological functions. Among them, two main functions are identified: light-driven ion transporters and sensory receptor for phototaxis responses. Bacteriorhodopsin belong to ion-transporter type and is the first and best-understood archaeal rhodopsin which can pump protons out of cell upon light illumination to induce a proton gradient, which further lead to
ATP synthesis via F1Fo ATP synthase system.
Haloquadratum walsbyi genome was completed in 2006 and a total of three retinal-binding proteins were predicted, including two bacteriorhodopsins-like and one halorhodopsin-like chloride pumping photoreceptor. The two opsin genes in H. walsbyi, bop1 and bop2, were those that identified and assumed to encode two bacteriorhodopsin-like proteins, and their protein products are named HwBR and MR, respectively. The goal of this study is to compare the features between HwBR and MR to further understand whether H. walsbyi indeed posses
a two-bacretiorhodopsin system.
The protein sequence alignment and phylogenetic tree analysis showed the high identity of HwBR and MR. The genes HwbopI and HwbopII were cloned and expressed in E. Coli C43(DE3) for biochemical property studies, and the maximum absorbance were 552 nm and 488 nm, respectively. The light-driven proton pumping activity showed that HwBR to have light-driven proton transportation function as that found in bacteriorhodopsin, while MR showed no such activity at all, even when being activated with the 470nm laser beam. The photocycle kinetic measurements showed HwBR to have a 200 msec photocycle time just like bacteriorhodopsin, while it was a 2-second for MR, a time course similar to those found in sensory rhodopsins. Further different mutagenesis studies at Asp85 and Asp96, two conserved and functionally critical residues found in bacteriorhodopsin, found only HwBR showed¬- the same impact in photocycle kinetics as those reported in HsBR but not in MR. The overall results in this study generated three conclusions: i) H. walsbyi does not contain a two-bacteriorhodopsin system as that identified in Haloarcula marismortui ; it has one bacteriorhodopsin-like protein and one unique rhodopsin, MR, ii) MR possesses biochemical properties that can only be classified as one that between ion-type and sensory-type. Further experiments are needed to perform to determine the function of MR. iii) The existence of a corresponding resiude Asp96 as in HsBR no longer clearly separates a bacteriorhodopsin-type
retinal-binding protein from a sensory rhodopsin-type.
Subjects
rhodopsin
bacteriorhodpsin
MR
photocycle
proton pump activity
Type
thesis
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