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

Chloride-dependent spectral changes in the absorption spectrum of HRs obtained from E. coli membraneThe spectral changes of HmHR (a) and HwHR (b); the corresponding difference spectra of HmHR (c) and HwHR (d); the absorbance change at 650 nm of HmHR (e) and HwHR (f) are shown, respectively. Purified HRs were incubated in the buffer solutions, which consisted of 50 mM Mes, pH 5.8, including 0.05% DDM and the appropriate amount of NaCl. In (a, b), the insert pictures show the purified HR, which was resuspended under the noted condition. In (a–d), the grey scale indicates the decrease of chloride concentrations. In (e, f), the absorbance changes were normalized and the solid line denotes the best-fitted model.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3475450&req=5

Figure 3: Chloride-dependent spectral changes in the absorption spectrum of HRs obtained from E. coli membraneThe spectral changes of HmHR (a) and HwHR (b); the corresponding difference spectra of HmHR (c) and HwHR (d); the absorbance change at 650 nm of HmHR (e) and HwHR (f) are shown, respectively. Purified HRs were incubated in the buffer solutions, which consisted of 50 mM Mes, pH 5.8, including 0.05% DDM and the appropriate amount of NaCl. In (a, b), the insert pictures show the purified HR, which was resuspended under the noted condition. In (a–d), the grey scale indicates the decrease of chloride concentrations. In (e, f), the absorbance changes were normalized and the solid line denotes the best-fitted model.

Mentions: The H. walsbyi cells were shown to tolerate 2 M MgCl2, an unusual environment for any other currently known HRs. To investigate this observation, the chloride-dependent spectral shifts of HwHR and HmHR were measured (Figure 3). The chloride-dependent spectral shift indicates the binding affinity of chloride ions to HRs, which is analogous to the binding behaviour of a ligand to a receptor, and it can be described via the Hill equation [21]. The results showed that, for HmHR (Figure 3a), an approximately 15 nm shift was observed in conditions of less than 1 M of chloride. The spectral shifts ceased at 591 nm when the concentration of NaCl fell below 1 mM, and the so-called ‘blue HR’ (Figure 3a, inset) was formed as observed in other studies [11,19]. HmHR had no spectral changes from 4 to 1 M NaCl. HwHR (Figure 3b), by contrast, showed no significant spectral shift throughout various chloride concentrations, with only a slight 2–3 nm of either redshifted or blueshifted changes observed in the range of 250 mM to 15 μM NaCl before finally settling at 576 nm, close to the ground state.


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 spectral changes in the absorption spectrum of HRs obtained from E. coli membraneThe spectral changes of HmHR (a) and HwHR (b); the corresponding difference spectra of HmHR (c) and HwHR (d); the absorbance change at 650 nm of HmHR (e) and HwHR (f) are shown, respectively. Purified HRs were incubated in the buffer solutions, which consisted of 50 mM Mes, pH 5.8, including 0.05% DDM and the appropriate amount of NaCl. In (a, b), the insert pictures show the purified HR, which was resuspended under the noted condition. In (a–d), the grey scale indicates the decrease of chloride concentrations. In (e, f), the absorbance changes were normalized and the solid line denotes the best-fitted model.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Chloride-dependent spectral changes in the absorption spectrum of HRs obtained from E. coli membraneThe spectral changes of HmHR (a) and HwHR (b); the corresponding difference spectra of HmHR (c) and HwHR (d); the absorbance change at 650 nm of HmHR (e) and HwHR (f) are shown, respectively. Purified HRs were incubated in the buffer solutions, which consisted of 50 mM Mes, pH 5.8, including 0.05% DDM and the appropriate amount of NaCl. In (a, b), the insert pictures show the purified HR, which was resuspended under the noted condition. In (a–d), the grey scale indicates the decrease of chloride concentrations. In (e, f), the absorbance changes were normalized and the solid line denotes the best-fitted model.
Mentions: The H. walsbyi cells were shown to tolerate 2 M MgCl2, an unusual environment for any other currently known HRs. To investigate this observation, the chloride-dependent spectral shifts of HwHR and HmHR were measured (Figure 3). The chloride-dependent spectral shift indicates the binding affinity of chloride ions to HRs, which is analogous to the binding behaviour of a ligand to a receptor, and it can be described via the Hill equation [21]. The results showed that, for HmHR (Figure 3a), an approximately 15 nm shift was observed in conditions of less than 1 M of chloride. The spectral shifts ceased at 591 nm when the concentration of NaCl fell below 1 mM, and the so-called ‘blue HR’ (Figure 3a, inset) was formed as observed in other studies [11,19]. HmHR had no spectral changes from 4 to 1 M NaCl. HwHR (Figure 3b), by contrast, showed no significant spectral shift throughout various chloride concentrations, with only a slight 2–3 nm of either redshifted or blueshifted changes observed in the range of 250 mM to 15 μM NaCl before finally settling at 576 nm, close to the ground state.

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