Solid State 15N NMR Evidence for a Complex Schiff Base Counterion in the Visual G-Protein-Coupled Receptor Rhodopsin

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Abstract
Using the baculovirus/Sf9 cell expression system, we have incorporated 99% 15N-enriched [α,ε-15N2]-l-lysine into the rod visual pigment rhodopsin. We have subsequently investigated the protonated Schiff base (pSB) linkage in the [α,ε-15N2]Lys−rhodopsin with cross-polarization magic angle spinning (CP/MAS) 15N NMR. The Schiff base (SB) 15N in [α,ε-15N2]Lys−rhodopsin resonates with an isotropic shift σI of 155.9 ppm, relative to 5.6 M 15NH4Cl. This suggests that the SB in rhodopsin is protonated and stabilized by a complex counterion. The 15N shifts of retinal SBs correlate with the energy difference between the ground and excited states and the frequency of maximum visible absorbance, νmax, associated with the π−π* transition of the polyene chromophore. Experimental modeling of the relation between the νmax and the size of the counterion with a set of pSBs provides strong evidence that the charged chromophore in rhodopsin is stabilized by a counterion with an estimated effective center−center distance (deff) between the counterion and the pSB of 0.43 ± 0.01 nm. While selected prokaryotic proteins and complexes have been labeled before, this is the first time to our knowledge that a 15N-labeled eukaryotic membrane protein has been generated in sufficient amount for such NMR investigations.