Limits...
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.

Show MeSH

Related in: MedlinePlus

Spectroscopic characterization and functional determination of HmHR and HwHRUV/Vis absorbance spectra of HmHR (a) and HwHR (b). The purified protein was resuspended in a buffer containing 50 mM Mes, 4 M NaCl, pH 5.8 and 0.05% DDM. The passive proton uptake activity of HmHR (c) and HwHR (d) expressed in E. coli is shown. The grey bar region indicates the sample incubated under the dark condition, and the illumination period was 100 s, then followed by another 100 s of dark incubation. The solid line shows the sample treated without CCCP, and the dotted line indicates the sample with CCCP. Laser-induced absorbance changes of HmHR (e) and HwHR (f) under 50 mM Mes, 4 M NaCl and 0.05% DDM, pH 5.8. The absorbance change was monitored at 580 nm. The data were analysed and fitted to one exponential decay (shown as a black line) for photocycle recovery half time.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3475450&req=5

Figure 2: Spectroscopic characterization and functional determination of HmHR and HwHRUV/Vis absorbance spectra of HmHR (a) and HwHR (b). The purified protein was resuspended in a buffer containing 50 mM Mes, 4 M NaCl, pH 5.8 and 0.05% DDM. The passive proton uptake activity of HmHR (c) and HwHR (d) expressed in E. coli is shown. The grey bar region indicates the sample incubated under the dark condition, and the illumination period was 100 s, then followed by another 100 s of dark incubation. The solid line shows the sample treated without CCCP, and the dotted line indicates the sample with CCCP. Laser-induced absorbance changes of HmHR (e) and HwHR (f) under 50 mM Mes, 4 M NaCl and 0.05% DDM, pH 5.8. The absorbance change was monitored at 580 nm. The data were analysed and fitted to one exponential decay (shown as a black line) for photocycle recovery half time.

Mentions: The UV/Vis spectra showed the λmax of HmHR to be 576 nm (Figure 2a), whereas the λmax of HwHR was 573 nm (Figure 2b). Compared with all well-known HRs, all of them were within approximately 3 nm of 575 nm (summarized in Supplementary Table S3 at http://www.bioscirep.org/bsr/032/bsr0320501add.htm).


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)

Spectroscopic characterization and functional determination of HmHR and HwHRUV/Vis absorbance spectra of HmHR (a) and HwHR (b). The purified protein was resuspended in a buffer containing 50 mM Mes, 4 M NaCl, pH 5.8 and 0.05% DDM. The passive proton uptake activity of HmHR (c) and HwHR (d) expressed in E. coli is shown. The grey bar region indicates the sample incubated under the dark condition, and the illumination period was 100 s, then followed by another 100 s of dark incubation. The solid line shows the sample treated without CCCP, and the dotted line indicates the sample with CCCP. Laser-induced absorbance changes of HmHR (e) and HwHR (f) under 50 mM Mes, 4 M NaCl and 0.05% DDM, pH 5.8. The absorbance change was monitored at 580 nm. The data were analysed and fitted to one exponential decay (shown as a black line) for photocycle recovery half time.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Spectroscopic characterization and functional determination of HmHR and HwHRUV/Vis absorbance spectra of HmHR (a) and HwHR (b). The purified protein was resuspended in a buffer containing 50 mM Mes, 4 M NaCl, pH 5.8 and 0.05% DDM. The passive proton uptake activity of HmHR (c) and HwHR (d) expressed in E. coli is shown. The grey bar region indicates the sample incubated under the dark condition, and the illumination period was 100 s, then followed by another 100 s of dark incubation. The solid line shows the sample treated without CCCP, and the dotted line indicates the sample with CCCP. Laser-induced absorbance changes of HmHR (e) and HwHR (f) under 50 mM Mes, 4 M NaCl and 0.05% DDM, pH 5.8. The absorbance change was monitored at 580 nm. The data were analysed and fitted to one exponential decay (shown as a black line) for photocycle recovery half time.
Mentions: The UV/Vis spectra showed the λmax of HmHR to be 576 nm (Figure 2a), whereas the λmax of HwHR was 573 nm (Figure 2b). Compared with all well-known HRs, all of them were within approximately 3 nm of 575 nm (summarized in Supplementary Table S3 at http://www.bioscirep.org/bsr/032/bsr0320501add.htm).

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