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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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Chloride-dependent spectra of various mutants of HwHR and A246S HsHRThe UV/Vis spectra of T186A HwHR (a), S262A HwHR (b) and A246S HsHR (c) were measured under different chloride concentrations to determine the chloride-dependent colour tuning. All purified proteins were dissolved in 50 mM Mes (pH 5.8) and 0.05% DDM buffer containing 4 M NaCl (black line) or 0 M NaCl (grey line).
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Figure 4: Chloride-dependent spectra of various mutants of HwHR and A246S HsHRThe UV/Vis spectra of T186A HwHR (a), S262A HwHR (b) and A246S HsHR (c) were measured under different chloride concentrations to determine the chloride-dependent colour tuning. All purified proteins were dissolved in 50 mM Mes (pH 5.8) and 0.05% DDM buffer containing 4 M NaCl (black line) or 0 M NaCl (grey line).

Mentions: As Thr186 and Ser262 are both typically conserved alanine residues in other known halobacterial HRs, single residue substitutions of T186A and S262A in HwHR were constructed. The S262A-HwHR eliminated the chloride-independent λmax change feature (Figure 4b), as a blueshift of approximately 8 nm under low chloride conditions was observed but T186A-HwHR (Figure 4a) behaved like the wild-type. However, the light-driven chloride-pumping capability and the photocycle of S262A were comparable with the wild-type (Supplementary Table S4 at http://www.bioscirep.org/bsr/032/bsr0320501add.htm). The chloride-binding affinity assay showed that the Kd was 10.40 mM for the S262A-HwHR, comparable with HsHR. The Hill coefficient (n) was 0.91, suggesting no significant change in chloride-binding from HwHR.


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)

Chloride-dependent spectra of various mutants of HwHR and A246S HsHRThe UV/Vis spectra of T186A HwHR (a), S262A HwHR (b) and A246S HsHR (c) were measured under different chloride concentrations to determine the chloride-dependent colour tuning. All purified proteins were dissolved in 50 mM Mes (pH 5.8) and 0.05% DDM buffer containing 4 M NaCl (black line) or 0 M NaCl (grey line).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Chloride-dependent spectra of various mutants of HwHR and A246S HsHRThe UV/Vis spectra of T186A HwHR (a), S262A HwHR (b) and A246S HsHR (c) were measured under different chloride concentrations to determine the chloride-dependent colour tuning. All purified proteins were dissolved in 50 mM Mes (pH 5.8) and 0.05% DDM buffer containing 4 M NaCl (black line) or 0 M NaCl (grey line).
Mentions: As Thr186 and Ser262 are both typically conserved alanine residues in other known halobacterial HRs, single residue substitutions of T186A and S262A in HwHR were constructed. The S262A-HwHR eliminated the chloride-independent λmax change feature (Figure 4b), as a blueshift of approximately 8 nm under low chloride conditions was observed but T186A-HwHR (Figure 4a) behaved like the wild-type. However, the light-driven chloride-pumping capability and the photocycle of S262A were comparable with the wild-type (Supplementary Table S4 at http://www.bioscirep.org/bsr/032/bsr0320501add.htm). The chloride-binding affinity assay showed that the Kd was 10.40 mM for the S262A-HwHR, comparable with HsHR. The Hill coefficient (n) was 0.91, suggesting no significant change in chloride-binding from HwHR.

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