Short and long range 2D ¹⁵N–¹⁵N NMR correlations among peptide groups by novel solid state dipolar mixing schemes

A recently developed homonuclear dipolar recoupling scheme, Adiabatic Linearly FREquency Swept reCOupling (AL FRESCO), was applied to record two-dimensional (2D) ¹⁵N–¹⁵N correlations on uniformly ¹⁵N-labeled GB1 powders. A major feature exploited in these ¹⁵N–¹⁵N correlations was AL FRESCO’s remarkably low RF power demands, which enabled seconds-long mixing schemes when establishing direct correlations. These ¹⁵N–¹⁵N mixing schemes proved efficient regardless of the magic-angle spinning (MAS) rate and, being nearly free from dipolar truncation effects, they enabled the detection of long-range, weak dipolar couplings, even in the presence of strong short-range dipolar couplings. This led to a connectivity information that was significantly better than that obtained with spontaneously proton-driven, ¹⁵N spin-diffusion experiments. An indirect approach producing long-range ¹⁵N–¹⁵N correlations was also tested, relying on short (ms-long) ¹H^N–¹H^N mixings schemes while applying AL FRESCO chirped pulses along the ¹⁵N channel. These indirect mixing schemes produced numerous long-distance N_i–N_i±n (n = 2 − 5) correlations, that might be useful for characterizing three-dimensional arrangements in proteins. Once again, these AL FRESCO mediated experiments proved more informative than variants based on spin-diffusion-based ¹H^N–¹H^N counterparts.