Two dimensional (2D) solid-state ¹³C...¹³C dipolar recoupling experiments are performed on a series of model compounds and on the visual pigment rhodopsin to establish the most effective method for long range distance measurements in reconstituted membrane proteins. The effects of uniform labeling, inhomogeneous B₁ fields, relaxation and dipolar truncation on cross peak intensity are investigated through NMR measurements of simple amino acid and peptide model compounds. We first show that dipolar assisted rotational resonance (DARR) is more effective than RFDR in recoupling long-range dipolar interactions in these model systems. We then use DARR to establish ¹³C-¹³C correlations in rhodopsin. In rhodopsin containing 4-¹³C-Tyr and 8,19-¹³C retinal, we observe two distinct tyrosine-to-retinal correlations in the DARR spectrum. The most intense cross peak arises from a correlation between Tyr268 and the retinal 19-¹³CH₃, which are 4.8 Å apart in the rhodopsin crystal structure. A second cross peak arises from a correlation between Tyr191 and the retinal 19-¹³CH₃, which are 5.5 Å apart in the crystal structure. These data demonstrate that long range ¹³C. . .¹³C correlations can be obtained in non-crystalline integral membrane proteins reconstituted into lipid membranes containing less than 150 nmoles of protein. In rhodopsin containing 2-¹³C Gly121 and U-¹³C Trp265, we do not observe a Trp-Gly cross peak in the DARR spectrum despite their close proximity (3.6 Å) in the crystal structure. Based on model compounds, the absence of a ¹³C. . .¹³C cross peak is due to loss of intensity in the diagonal Trp resonances rather than to dipolar truncation.

DARR - dipolar assisted rotational resonance; GPCR - G protein-coupled receptor; MAS - magic angle spinning; NMR - nuclear magnetic resonance; PDSD - proton-driven spin diffusion; REDOR - rotational echo double resonance; RFDR - radiofrequency driven recoupling; TM - transmembrane; VACP - variable amplitude cross polarization