Solid state N-15 NMR evidence for a complex Schiff base counterion in the visual G-protein-coupled receptor rhodopsin

Using the baculovirus/Sf9 cell expression system, we have incorporated 99% N-15-enriched [alpha,epsilon-N-15(2)]-L-lysine into the rod visual pigment rhodopsin. We have subsequently investigated the protonated Schiff base (pS B) linkage in the [alpha,epsilon-N-15(2)]Lys -rhodopsin with cross-polariza tion magic angle spinning (CP/MAS) N-15 NMR. The Schiff base (SB) N-15 in [ alpha,epsilon-N-15(2)]Lys-rhodopsin resonates with an isotropic shift at of 155.9 ppm, relative to 5.6 M (NH4Cl)-N-15. This suggests that the SE in rh odopsin is protonated and stabilized by a complex counterion. The N-15 shif ts Of retinal SBs correlate with the energy difference between the ground a nd excited states and the frequency of maximum visible absorbance, v(max), associated with the pi-pi* transition of the polyene chromophore, Experimen tal modeling of the relation between the v(max) and the size of the counter ion with a set of pSBs provides strong evidence that the charged chromophor e in rhodopsin is stabilized by a counterion with an estimated effective ce nter-center distance (d(eff)) between the counterion and the pSB of 0.43 +/ - 0.01 nm. While selected prokaryotic proteins and complexes have been labe led before, this is the first time to our knowledge that a N-15-labeled euk aryotic membrane protein has been generated in sufficient amount for such N MR investigations.