Transverse relaxation optimized 3D and 4D 15N/15N separated NOESY experiments of 15N labeled proteins

Distance constraints are an important complement to orientational constraints. While a high-resolution monomer structure of the ion channel forming polypeptide, gramicidin A, has been solved with 120 orientational constraints, the precise geometry of the dimer interface has not been characterized. Here, using both ¹³C and ¹⁵N labeled gramicidin A samples in hydrated phospholipid bilayers, both inter- and intramolecular distances have been measured with a recently developed simultaneous frequency and amplitude modulation (SFAM) solid-state NMR scheme. Using this approach ¹⁵N-¹³C₁ residual dipolar couplings across a hydrogen bond as small as 20 ± 2 Hz have been characterized. While such distances are on the order of 4.2 ± 0.2 Å, the spectroscopy is complicated by rapid global motion of the molecular structure about the bilayer normal and channel axis. Consequently, the nominal 40 Hz dipolar coupling is averaged depending on the orientation of the internuclear vector with respect to the motional axis. The intermolecular distance confirmed the previously described monomeric structure, while the intramolecular distance across the monomer–monomer interface defined this junction and confirmed the previous model of this interface.