Solid-state 19F-NMR analysis of 19F-labeled tryptophan in gramicidin A in oriented membranes

The response of membrane-associated peptides toward the lipid environment or other binding partners can be monitored by solid-state NMR of suitably labeled side chains. Tryptophan is a prominent amino acid in transmembrane helices, and its F-19-labeled analogues are generally biocompatible and cause little structural perturbation. Hence, we use 5F-Trp as a highly sensitive NMR probe to monitor the conformation and dynamics of the indole ring. To establish this F-19-NMR strategy, gramicidin A was labeled with 5F-Trp in position 13 or 15, whose (chi1)/(chi2) torsion angles are known from previous H-2-NMR studies. First, the alignment of the F-19 chemical shift anisotropy tensor within the membrane was deduced by lineshape analysis of oriented samples. Next, the three principal axes of the F-19 chemical shift anisotropy tensor were assigned within the molecular frame of the indole ring. Finally, determination of (chi1)/(chi2) for 5F-Trp in the lipid gel phase showed that the side chain alignment differs by up to 20degrees from its known conformation in the liquid crystalline state. The sensitivity gain of F-19-NMR and the reduction in the amount of material was at least 10-fold compared with previous H-2-NMR studies on the same system and 100-fold compared with N-15-NMR.