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Ser(262) determines the chloride-dependent colour tuning of a new halorhodopsin from Haloquadratum walsbyi.

Fu HY, Chang YN, Jheng MJ, Yang CS - Biosci. Rep. (2012)

Bottom Line: The results showed them to have similar properties to two HRs reported previously.A Ser262 to alanine replacement in HwHR eliminated the chloride-independent colour tuning, whereas an Ala246 to serine mutagenesis in HsHR transformed it to have chloride-independent colour tuning similar to that of HwHR.Thus Ser262 is a key residue for the mechanism of chloride-dependent colour tuning in HwHR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Light is an important environmental signal for all organisms on earth because it is essential for physiological signalling and the regulation of most biological systems. Halophiles found in salt-saturated ponds encode various archaeal rhodopsins and thereby harvest various wavelengths of light either for ion transportation or as sensory mediators. HR (halorhodopsin), one of the microbial rhodopsins, senses yellow light and transports chloride or other halides into the cytoplasm to maintain the osmotic balance during cell growth, and it exists almost ubiquitously in all known halobacteria. To date, only two HRs, isolated from HsHR (Halobacterium salinarum HR) and NpHR (Natronomonas pharaonis HR), have been characterized. In the present study, two new HRs, HmHR (Haloarcula marismortui HR) and HwHR (Haloquadratum walsbyi HR), were functionally overexpressed in Escherichia coli, and the maximum absorbance (λmax) of the purified proteins, the light-driven chloride uptake and the chloride-binding affinity were measured. The results showed them to have similar properties to two HRs reported previously. However, the λmax of HwHR is extremely consistent in a wide range of salt/chloride concentrations, which had not been observed previously. A structural-based sequence alignment identified a single serine residue at 262 in HwHR, which is typically a conserved alanine in all other known HRs. A Ser262 to alanine replacement in HwHR eliminated the chloride-independent colour tuning, whereas an Ala246 to serine mutagenesis in HsHR transformed it to have chloride-independent colour tuning similar to that of HwHR. Thus Ser262 is a key residue for the mechanism of chloride-dependent colour tuning in HwHR.

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Related in: MedlinePlus

Bioinformatic analysis of microbial HRs and structural alignment of HsHR and HwHR(a) Phylogenetic tree of 17 annotated microbial HRs. The green-underlined organisms are the sources of the two HRs from bacteria. The red rectangles indicate the two well-studied HRs from Halobacterium salinarum and Natronobacterium pharaonis; the blue rectangles indicate the two HRs from HmHR and HwHR in the present study. (b) Amino acid sequence alignment of critical residues involved in chloride translocation and of interest in the present study. The first two rows are numbered with the atomic resolution structures based on NpHR (PDB ID: 3A7K) and HsHR (PDB ID: 1E12). The black rectangles indicate the two HRs in the present study. The shadowed grey scale marks the conserved residues, and the critical spectral tuner one is annotated with the black spot. (c) Structural information of HsHR (grey, PDB ID: 1E12) and HwHR (red, modelled by SWISS-MODEL). The conserved chloride transport residues (stick) are labelled with their name and number (Thr66, His95, Arg108, Thr111, Ala127, Arg161, Arg200, Thr203, Glu219 and Asp238). The yellow stick is all-trans retinal; the conserved residues Thr111 and Asp238 harbour a chloride ion (green sphere) in the structures of HsHR and HwHR. The residues (cyan stick) indicate the mutated site of HwHR in this study. The top is the periplasmic region and the bottom is the cytoplasm. The 90o rotation is for the bottom view for both HRs.
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Figure 1: Bioinformatic analysis of microbial HRs and structural alignment of HsHR and HwHR(a) Phylogenetic tree of 17 annotated microbial HRs. The green-underlined organisms are the sources of the two HRs from bacteria. The red rectangles indicate the two well-studied HRs from Halobacterium salinarum and Natronobacterium pharaonis; the blue rectangles indicate the two HRs from HmHR and HwHR in the present study. (b) Amino acid sequence alignment of critical residues involved in chloride translocation and of interest in the present study. The first two rows are numbered with the atomic resolution structures based on NpHR (PDB ID: 3A7K) and HsHR (PDB ID: 1E12). The black rectangles indicate the two HRs in the present study. The shadowed grey scale marks the conserved residues, and the critical spectral tuner one is annotated with the black spot. (c) Structural information of HsHR (grey, PDB ID: 1E12) and HwHR (red, modelled by SWISS-MODEL). The conserved chloride transport residues (stick) are labelled with their name and number (Thr66, His95, Arg108, Thr111, Ala127, Arg161, Arg200, Thr203, Glu219 and Asp238). The yellow stick is all-trans retinal; the conserved residues Thr111 and Asp238 harbour a chloride ion (green sphere) in the structures of HsHR and HwHR. The residues (cyan stick) indicate the mutated site of HwHR in this study. The top is the periplasmic region and the bottom is the cytoplasm. The 90o rotation is for the bottom view for both HRs.

Mentions: HRs are prevalent in Halobacteria; more than 15 strains (Supplementary Table S1) of Halobacteria have been completely sequenced to date, and at least nine of these were proven or predicted to have hop genes. A phylogenic tree (Figure 1a) and sequence alignment (Figure 1b, and Supplementary Figure S1 at http://www.bioscirep.org/bsr/032/bsr0320501add.htm) of 17 annotated HRs showed high sequence identity (55–68%) among them, and the previously reported key residues involved in chloride transportation, i.e. Arg108/Thr111 and Arg200/Thr203 in HsHR [18], were found to be conserved in all HRs. Although the classification of HRs were not correlated with the evolution [29], HmHR and HwHR, which were the focus in the present study, showed higher sequence similarity with NpHR and HsHR respectively.


Ser(262) determines the chloride-dependent colour tuning of a new halorhodopsin from Haloquadratum walsbyi.

Fu HY, Chang YN, Jheng MJ, Yang CS - Biosci. Rep. (2012)

Bioinformatic analysis of microbial HRs and structural alignment of HsHR and HwHR(a) Phylogenetic tree of 17 annotated microbial HRs. The green-underlined organisms are the sources of the two HRs from bacteria. The red rectangles indicate the two well-studied HRs from Halobacterium salinarum and Natronobacterium pharaonis; the blue rectangles indicate the two HRs from HmHR and HwHR in the present study. (b) Amino acid sequence alignment of critical residues involved in chloride translocation and of interest in the present study. The first two rows are numbered with the atomic resolution structures based on NpHR (PDB ID: 3A7K) and HsHR (PDB ID: 1E12). The black rectangles indicate the two HRs in the present study. The shadowed grey scale marks the conserved residues, and the critical spectral tuner one is annotated with the black spot. (c) Structural information of HsHR (grey, PDB ID: 1E12) and HwHR (red, modelled by SWISS-MODEL). The conserved chloride transport residues (stick) are labelled with their name and number (Thr66, His95, Arg108, Thr111, Ala127, Arg161, Arg200, Thr203, Glu219 and Asp238). The yellow stick is all-trans retinal; the conserved residues Thr111 and Asp238 harbour a chloride ion (green sphere) in the structures of HsHR and HwHR. The residues (cyan stick) indicate the mutated site of HwHR in this study. The top is the periplasmic region and the bottom is the cytoplasm. The 90o rotation is for the bottom view for both HRs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475450&req=5

Figure 1: Bioinformatic analysis of microbial HRs and structural alignment of HsHR and HwHR(a) Phylogenetic tree of 17 annotated microbial HRs. The green-underlined organisms are the sources of the two HRs from bacteria. The red rectangles indicate the two well-studied HRs from Halobacterium salinarum and Natronobacterium pharaonis; the blue rectangles indicate the two HRs from HmHR and HwHR in the present study. (b) Amino acid sequence alignment of critical residues involved in chloride translocation and of interest in the present study. The first two rows are numbered with the atomic resolution structures based on NpHR (PDB ID: 3A7K) and HsHR (PDB ID: 1E12). The black rectangles indicate the two HRs in the present study. The shadowed grey scale marks the conserved residues, and the critical spectral tuner one is annotated with the black spot. (c) Structural information of HsHR (grey, PDB ID: 1E12) and HwHR (red, modelled by SWISS-MODEL). The conserved chloride transport residues (stick) are labelled with their name and number (Thr66, His95, Arg108, Thr111, Ala127, Arg161, Arg200, Thr203, Glu219 and Asp238). The yellow stick is all-trans retinal; the conserved residues Thr111 and Asp238 harbour a chloride ion (green sphere) in the structures of HsHR and HwHR. The residues (cyan stick) indicate the mutated site of HwHR in this study. The top is the periplasmic region and the bottom is the cytoplasm. The 90o rotation is for the bottom view for both HRs.
Mentions: HRs are prevalent in Halobacteria; more than 15 strains (Supplementary Table S1) of Halobacteria have been completely sequenced to date, and at least nine of these were proven or predicted to have hop genes. A phylogenic tree (Figure 1a) and sequence alignment (Figure 1b, and Supplementary Figure S1 at http://www.bioscirep.org/bsr/032/bsr0320501add.htm) of 17 annotated HRs showed high sequence identity (55–68%) among them, and the previously reported key residues involved in chloride transportation, i.e. Arg108/Thr111 and Arg200/Thr203 in HsHR [18], were found to be conserved in all HRs. Although the classification of HRs were not correlated with the evolution [29], HmHR and HwHR, which were the focus in the present study, showed higher sequence similarity with NpHR and HsHR respectively.

Bottom Line: The results showed them to have similar properties to two HRs reported previously.A Ser262 to alanine replacement in HwHR eliminated the chloride-independent colour tuning, whereas an Ala246 to serine mutagenesis in HsHR transformed it to have chloride-independent colour tuning similar to that of HwHR.Thus Ser262 is a key residue for the mechanism of chloride-dependent colour tuning in HwHR.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Science and Technology, College of Life Science, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Light is an important environmental signal for all organisms on earth because it is essential for physiological signalling and the regulation of most biological systems. Halophiles found in salt-saturated ponds encode various archaeal rhodopsins and thereby harvest various wavelengths of light either for ion transportation or as sensory mediators. HR (halorhodopsin), one of the microbial rhodopsins, senses yellow light and transports chloride or other halides into the cytoplasm to maintain the osmotic balance during cell growth, and it exists almost ubiquitously in all known halobacteria. To date, only two HRs, isolated from HsHR (Halobacterium salinarum HR) and NpHR (Natronomonas pharaonis HR), have been characterized. In the present study, two new HRs, HmHR (Haloarcula marismortui HR) and HwHR (Haloquadratum walsbyi HR), were functionally overexpressed in Escherichia coli, and the maximum absorbance (λmax) of the purified proteins, the light-driven chloride uptake and the chloride-binding affinity were measured. The results showed them to have similar properties to two HRs reported previously. However, the λmax of HwHR is extremely consistent in a wide range of salt/chloride concentrations, which had not been observed previously. A structural-based sequence alignment identified a single serine residue at 262 in HwHR, which is typically a conserved alanine in all other known HRs. A Ser262 to alanine replacement in HwHR eliminated the chloride-independent colour tuning, whereas an Ala246 to serine mutagenesis in HsHR transformed it to have chloride-independent colour tuning similar to that of HwHR. Thus Ser262 is a key residue for the mechanism of chloride-dependent colour tuning in HwHR.

Show MeSH
Related in: MedlinePlus