Figure 5.

¹³C MAS difference spectra of rhodopsin, Meta I and Meta II. Difference spectra are shown for rhodopsin - Meta I (orange) and rhodopsin - Meta II (black) using rhodopsin isotopically labeled with ¹³Cζ-tyrosine, ¹³Cε-methionine, ¹³Cα-glycine and ¹³Cβ-cysteine. Positive peaks correspond to rhodopsin and negative peaks correspond to Meta I or Meta II. (A) The tyrosine ¹³Cζ difference spectrum between rhodopsin and Meta II exhibits two positive and two negative resonances. The two Meta II resonances at 158.9 and 153.1 ppm have been assigned to Tyr191^EL2 and Tyr206^5.41, respectively.²² These resonances are not observed in the rhodopsin - Meta I difference spectrum. (B) The methionine ¹³Cε difference spectrum between rhodopsin and Meta II exhibits three distinct positive and two negative resonances. The resonances associated with Met44 and Met288 have been assigned in both rhodopsin and Meta II.²² The Met257 resonance does not change between rhodopsin and Meta II. A strong negative cross peak is observed at 15.8 ppm and assigned to Met257 in Meta I. (C) The glycine ¹³Cα difference spectrum exhibits two positive and two negative resonances. The upfield resonances in rhodopsin at 41.5 ppm and in Meta II at 42.9 ppm have been assigned to Gly188^EL2.²² The appearance of a negative resonance at the position of Gly188^EL2 is not observed in the rhodopsin - Meta I difference spectrum. (D) In the cysteine ¹³Cβ difference spectrum between rhodopsin and Meta II, the chemical shift of Cys187^EL2 changes from 46.8 ppm to 50.1 ppm.²² The disulfide bridge between Cys187^EL2 and Cys110^3.25 tethers EL2 to the helical bundle.