Solid-State 19F-NMR Analysis of 19F-Labeled Tryptophan in Gramicidin A in Oriented Membranes

Abstract

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 19F-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 19F-NMR strategy, gramicidin A was labeled with 5F-Trp in position 13 or 15, whose χ 1/ χ 2 torsion angles are known from previous 2H-NMR studies. First, the alignment of the 19F chemical shift anisotropy tensor within the membrane was deduced by lineshape analysis of oriented samples. Next, the three principal axes of the 19F chemical shift anisotropy tensor were assigned within the molecular frame of the indole ring. Finally, determination of χ 1/ χ 2 for 5F-Trp in the lipid gel phase showed that the side chain alignment differs by up to 20° from its known conformation in the liquid crystalline state. The sensitivity gain of 19F-NMR and the reduction in the amount of material was at least 10-fold compared with previous 2H-NMR studies on the same system and 100-fold compared with 15N-NMR.