Probing the proton channel and the retinal binding site of Natronobacterium pharaonis sensory rhodopsin II

Biophysical Journal
Johann P KlareM Engelhard

Abstract

The sensory rhodopsin II from Natronobacterium pharaonis (NpSRII) was mutated to try to create functional properties characteristic of bacteriorhodopsin (BR), the proton pump from Halobacterium salinarum. Key residues from the cytoplasmic and extracellular proton transfer channel of BR as well as from the retinal binding site were chosen. The single site mutants L40T, F86D, P183E, and T204A did not display altered function as determined by the kinetics of their photocycles. However, the photocycle of each of the subsequent multisite mutations L40T/F86D, L40T/F86D/P183E, and L40T/F86D/P183E/T204A was quite different from that of the wild-type protein. The reprotonation of the Schiff base could be accelerated approximately 300- to 400-fold, to approximately two to three times faster than the corresponding reaction in BR. The greatest effect is observed for the quadruple mutant in which Thr-204 is replaced by Ala. This result indicates that mutations affecting conformational changes of the protein might be of decisive importance for the creation of BR-like functional properties.

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Citations

Aug 7, 2002·Current Opinion in Structural Biology·John L Spudich, Hartmut Luecke
Jul 31, 2010·Journal of Biomedicine & Biotechnology·Hideki KandoriYuji Furutani
Aug 16, 2011·Journal of Molecular Biology·Ivan GushchinValentin Gordeliy
Dec 3, 2014·Journal of Photochemistry and Photobiology. B, Biology·Jun TamogamiNaoki Kamo
Jun 5, 2013·Biochimica Et Biophysica Acta·Keiichi InoueYuki Sudo
Sep 3, 2013·Biochimica Et Biophysica Acta·Mathias GrotePeter Hegemann
Mar 9, 2019·Photochemistry and Photobiology·Wageiha MosslehyHeinz-Jürgen Steinhoff
Jun 25, 2003·Biochemistry·Kazumi ShimonoHideki Kandori

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