Solid-state NMR for the characterization of retinal chromophore and Schiff base in TAT rhodopsin embedded in membranes under weakly acidic conditions.

TAT rhodopsin extracted from the marine bacterium SAR11 HIMB114 has a characteristic Thr-Ala-Thr motif and contains both protonated and deprotonated states of Schiff base at physiological pH conditions due to the low p K a. Here, using solid-state NMR spectroscopy, we investigated the 13 C and 15 N NMR signals of retinal in only the protonated state of TAT in the 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine/1-palmitoyl-2-oleoyl- sn -glycero-3-phospho (1'-rac-glycerol) (POPE/POPG) membrane at weakly acidic conditions. In the 13 C NMR spectrum of 13 C retinal-labeled TAT rhodopsin, the isolated 14-13 C signals of 13- trans /15- anti and 13- cis /15- syn isomers were observed at a ratio of 7:3. 15 N retinal protonated Schiff base (RPSB) had a significantly higher magnetic field resonance at 160 ppm. In 15 N RPSB/λmax analysis, the plot of TAT largely deviated from the trend based on the retinylidene-halide model compounds and microbial rhodopsins. Our findings indicate that the RPSB of TAT forms a very weak interaction with the counterion.