Soun Kanada

Crystal structures of an O-like blue form and an anion-free yellow form of pharaonis halorhodopsin

Halorhodopsin from Natronomonas pharaonis (pHR) was previously crystallized into a monoclinic space group C2, and the structure of the chloride-bound purple form was determined. Here, we report the crystal structures of two chloride-free forms of pHR, that is, an O-like blue form and an M-like yellow form. When the C2 crystal was soaked in a chloride-free alkaline solution, the protein packing was largely altered and the yellow form containing all-trans retinal was generated. Upon neutralization, this yellow form was converted into the blue form. From structural comparison of the different forms of pHR, it was shown that the removal of a chloride ion from the primary binding site (site I), which is located between the retinal Schiff base and Thr126, is accompanied by such a deformation of helix C that the side chain of Thr126 moves toward helix G, leading to a significant shrinkage of site I. A large structural change is also induced in the chloride uptake pathway, where a flip motion of the side chain of Glu234 is accompanied by large movements of the surrounding aromatic residues. Irrespective of different charge distributions at the active site, there was no large difference in the structures of the yellow form and the blue form. It is shown that the yellow-to-purple transition is initiated by the entrance of one water and one HCl to the active site, where the proton and the chloride ion in HCl are transferred to the Schiff base and site I, respectively.

Large Deformation of Helix F during the Photoreaction Cycle of Pharaonis Halorhodopsin in Complex with Azide

Halorhodopsin from Natronomonas pharaonis (pHR), a retinylidene protein that functions as a light-driven chloride ion pump, is converted into a proton pump in the presence of azide ion. To clarify this conversion, we investigated light-induced structural changes in pHR using a C2 crystal that was prepared in the presence of Cl(-) and subsequently soaked in a solution containing azide ion. When the pHR-azide complex was illuminated at pH 9, a profound outward movement (∼4 Å) of the cytoplasmic half of helix F was observed in a subunit with the EF loop facing an open space. This movement created a long water channel between the retinal Schiff base and the cytoplasmic surface, along which a proton could be transported. Meanwhile, the middle moiety of helix C moved inward, leading to shrinkage of the primary anion-binding site (site I), and the azide molecule in site I was expelled out to the extracellular medium. The results suggest that the cytoplasmic half of helix F and the middle moiety of helix C act as different types of valves for active proton transport.

Structure of archaerhodopsin‐2 at 1.8 Å resolution

The same hexagonal array of archaerhodpsin-2 trimers as observed in the native membrane is reconstituted in a three-dimensional crystal prepared by the membrane fusion method. In this crystal, a pair of conserved glutamate residues in the proton-release pathway is maintained by a low-barrier hydrogen bond.