Ultra-high-field MAS NMR assay of a multispin labeled ligand bound to its G-protein receptor target in the natural membrane environment: Electronic structure of the retinylidene chromophore in rhodopsin

11-Z-[8,9,10,11,12, 13,14,15,19,20-C-13(10)]Retinal prepared by total synth esis is reconstituted with opsin to form rhodopsin in the natural lipid mem brane environment. The C-13 shifts are assigned with magic angle spinning N MR dipolar correlation spectroscopy in a single experiment and compared wit h data of singly labeled retinylidene ligands in detergent-solubilized rhod opsin. The use of multispin labeling in combination with 2-D correlation sp ectroscopy improves the relative accuracy of the shift measurements. We hav e used the chemical shift data to analyze the electronic structure of the r etinylidene ligand at three levels of understanding: (i) by specifying inte ractions between the C-13-labeled ligand and the G-protein-coupled receptor target, (ii) by making a charge assessment of the protonation of the Schif f base in rhodopsin, and (iii) by evaluating the total charge on the carbon s of the retinylidene chromophore. In this way it is shown that a conjugati on defect is the predominant ground-state property governing the molecular electronics of the retinylidene chromophore in rhodopsin. The cumulative ch emical shifts at the odd-numbered carbons (Delta sigma (odd)) Of 11-Z-proto nated Schiff base models relative to the unprotonated Schiff base can be us ed to measure the extent of delocalization of positive charge into the poly ene. For a series of 11-Z-protonated Schiff base models and rhodopsin, Delt a sigma (odd) appears to correlate linearly with the frequency of maximum v isible absorption. Since rhodopsin has the largest value of Delta sigma (od d), the data contribute to existing and converging spectroscopic evidence f or a complex counterion stabilizing the protonated Schiff base in the bindi ng pocket.