Spin labeling of Natronomonas pharaonis halorhodopsin: probing the cysteine residues environment

The Journal of Physical Chemistry. B
Keren Mevorat-KaplanMordechai Sheves

Abstract

Halorhodopsin from Natronomonas pharaonis (pHR) is a light-driven chloride pump that transports a chloride anion across the plasma membrane following light absorption by a retinal chromophore which initiates a photocycle. Analysis of the amino acid sequence of pHR reveals three cysteine residues (Cys160, Cys184, and Cys186) in helices D and E. Here we have labeled the cysteine residues with nitroxide spin labels and studied using electron paramagnetic resonance (EPR) spectroscopy their mobility, accessibility to various reagents, and the distance between the labels. It was revealed by following the d(1)/d parameter that the distance between the spin labels is ca. 13-15 Angstrom. The EPR spectrum suggests that one label has a restricted mobility while the other two are more mobile. Only one label is accessible to hydrophilic paramagnetic broadening reagents leading to the conclusion that this label is exposed to the water phase. All three labels are reduced by ascorbic acid and reoxidized by molecular oxygen. The rate of the oxidation is accelerated following retinal irradiation indicating that the protein experiences conformation alterations in the vicinity of the labels during the pigment photocycle. It is suggested that Cys18...Continue Reading

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Citations

May 1, 2021·Biochimica Et Biophysica Acta. Biomembranes·Hasin FerozManish Kumar
Oct 18, 2012·The Journal of Physical Chemistry Letters·Yuji FurutaniHideki Kandori

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