Structure and Heterogeneity of Retinal Chromophore in Chloride Pump Rhodopsins Revealed by Raman Optical Activity.

Chloride transport by microbial rhodopsins is actively being researched to understand how light energy is converted to drive ion pumping across cell membranes. Chloride pumps have been identified in archaea and eubacteria, and there are similarities and differences in the active site structures between these groups. Thus, it has not been clarified whether a common mechanism underlies the ion pump processes for all chloride-pumping rhodopsins. Here, we applied Raman optical activity (ROA) spectroscopy to two chloride pumps, Nonlabens marinus rhodopsin-3 (NM-R3) and halorhodopsin from the cyanobacterium Mastigocladopsis repens (MrHR). ROA is a vibrational spectroscopy that provides chiral sensitivity, and the sign of ROA signals can reveal twisting of cofactor molecules within proteins. Our ROA analysis revealed that the retinal Schiff base NH group orients toward the C helix and forms a direct hydrogen bond with a nearby chloride ion in NM-R3. In contrast, MrHR is suggested to contain two retinal conformations twisted in opposite directions; one conformation has a hydrogen bond with a chloride ion like NM-R3, while the other forms a hydrogen bond with a water molecule anchored by a G helix residue. These results suggest a general pump mechanism in which the chloride ion is "dragged" by the flipping Schiff base NH group upon photoisomerization.

"Watching" a Molecular Twist in a Protein by Raman Optical Activity.

Light-absorbing chromophores in photoreceptors contain a π-electron system and are intrinsically planar molecules. However, within a protein environment these cofactors often become nonplanar and chiral in a manner that is widely believed to be functionally important. When the same chromophore is out-of-plane distorted in opposite directions in different members of a protein family, such conformers become a set of enantiomers. In techniques using chiral optical spectroscopy such as Raman optical activity (ROA), such proteins are expected to show opposite signs in their spectra. Here we use two microbial rhodopsins, Gloeobacter rhodopsin and sodium ion pump rhodopsin (NaR), to provide the first experimental and theoretical evidence that the twist direction of the retinal chromophore indeed determines the sign of the ROA spectrum. We disrupt the hydrogen bond responsible for the distortion of the retinal in NaR and show that the sign of the ROA signals of this nonfunctional mutant is flipped. The reported ROA spectra are monosignate, a property that has been seen for a variety of photoreceptors, which we attribute to an energetically favorable gradual curvature of the chromophore.