Functional roles of tyrosine 185 during the bacteriorhodopsin photocycle as revealed by in situ spectroscopic studies.
Ding X., Sun C., Cui H., Chen S., Gao Y., Yang Y., Wang J., He X., Iuga D., Tian F., Watts A., Zhao X.
Tyrosine 185 (Y185), one of the aromatic residues within the retinal (Ret) chromophore binding pocket in helix F of bacteriorhodopsin (bR), is highly conserved among the microbial rhodopsin family proteins. Many studies have investigated the functions of Y185, but its underlying mechanism during the bR photocycle remains unclear. To address this research gap, in situ two-dimensional (2D) magic-angle spinning (MAS) solid-state NMR (ssNMR) of specifically labelled bR, combined with light-induced transient absorption change measurements, dynamic light scattering (DLS) measurements, titration analysis and site-directed mutagenesis, was used to elucidate the functional roles of Y185 during the bR photocycle in the native membrane environment. Different interaction modes were identified between Y185 and the Ret chromophore in the dark-adapted (inactive) state and M (active) state, indicating that Y185 may serve as a rotamer switch maintaining the protein dynamics, and plays an important role in the efficient proton-pumping mechanism in the bR purple membrane.