Spectral tuning in bacteriorhodopsin in the absence of counterion and coplanarization effects.

The basis for wavelength regulation in bacteriorhodopsin (BR) and retinylidene proteins in general has been studied for decades but is still only partially understood. Here we report the preparation and spectroscopic characterization of BR analogs aimed at investigating the existence of spectral tuning mechanisms other than the two widely accepted mechanisms, weakened counterion interactions and ring/chain coplanarization. We synthesized two novel retinal analogs containing a saturated 13-14 bond, which interrupts the interaction of the protein counterions with the chromophore conjugation system. Furthermore, one of the analogs has a planar polyene system so that the contribution to the red shift of BR by retinal ring/chain coplanarization is also absent. We incorporated these analogs into bacterioopsin and discovered a sizable amount of red shift, which can be accounted for by interactions between the polar or polarizable groups of the protein and the retinal polyene chain. Our results suggest that the wavelength regulation in BR is achieved by synergistic chromophore/protein interactions including ring/chain coplanarization, excited state stabilization by polar or polarizable protein side chains located along the polyene chain, and weakened counterion interactions near the Schiff base positive charge.