Strongly Hydrogen Bonded Water Networks Detected in the Archaerhodopsin-3 Proton Pump

Abstract

We examined structural changes of the protein, retinal chromophore and internal water molecules during the photocycle of Archaerhodopsin-3 (AR3), a light-driven proton pump found in Halobrubrum sodemense. This protein, which belongs to the archael rhodopsin family, has a ∼75% sequence homology with the more extensively studied bacteriorhodopsin (BR) proton pump from Halobacterium salinarum. Recent interest has focused on AR3 because of its ability to serve both as a high-performance genetically targetable optical silencer of neuronal activity and as a membrane voltage sensor. Low-temperature and rapid-scan time-resolved FTIR-difference spectroscopy revealed that the conformational changes during formation of the K, M and N photocycle intermediates are similar, although not identical, to BR. Positive/negative bands in the region above 3600 cm-1 which have previously been assigned to structural changes of weakly hydrogen bonded internal water molecules were substantially different between AR3 and BR. This included the absence of positive bands recently associated with a chain of proton transporting water molecules in the cytoplasmic channel and a weakly hydrogen bonded water (W401) which is part of a hydrogen-bonded pentagonal cluster located near the retinal Schiff base. However, many of the broad IR continuum changes below 3000 cm-1 assigned to networks of water molecules involved in proton transport through cytoplasmic and extracellular portions of the protein were very similar in AR3 and BR.