Sensitivity enhanced 14N/14N correlations to probe inter-beta-sheet interactions using fast magic angle spinning solid-state NMR in biological solids

Abstract

¹⁴N/¹⁴N correlations are vital for structural studies of solid samples, especially those in which ¹⁵N isotopic enrichment is challenging, time-consuming and expensive. Although ¹⁴N nuclei have high isotopic abundance (99.6%), there are inherent difficulties in observing ¹⁴N/¹⁴N correlations due to limited resolution and sensitivity related to: (i) low ¹⁴N gyromagnetic ratio (γ), (ii) large ¹⁴N quadrupolar couplings, (iii) integer ¹⁴N spin quantum number (I = 1), and (iv) very weak ¹⁴N–¹⁴N dipolar couplings. Previously, we demonstrated a proton-detected 3D ¹⁴N/¹⁴N/¹H correlation experiment at fast magic angle spinning (MAS) on L-histidine·HCl·H₂O utilizing a through-bond (J) and residual dipolar-splitting (RDS) based heteronuclear multiple quantum correlation (J-HMQC) sequence mediated through ¹H/¹H radio-frequency driven recoupling (RFDR). As an extension of our previous work, in this study we show the utility of dipolar-based HMQC (D-HMQC) in combination with ¹H/¹H RFDR mixing to obtain sensitivity enhanced ¹⁴N/¹⁴N correlations in more complex biological solids such as a glycyl-L-alanine (Gly-L-Ala) dipeptide, and parallel (P) and antiparallel (AP) β-strand alanine tripeptides (P-(Ala)₃ and AP-(Ala)₃, respectively). These systems highlight the mandatory necessity of 3D ¹⁴N/¹⁴N/¹H measurements to get ¹⁴N/¹⁴N correlations when the amide proton resonances are overlapped. Moreover, the application of long selective ¹⁴N pulses, instead of short hard ones, is shown to improve the sensitivity. Globally, we demonstrate that replacing J-scalar with dipolar interaction and hard- with selective-¹⁴N pulses allows gaining a factor of ca. 360 in experimental time. On the basis of intermolecular NH/NH distances and ¹⁴N quadrupolar tensor orientations, ¹⁴N/¹⁴N correlations are effectively utilized to make a clear distinction between the parallel and antiparallel arrangements of the β-strands in (Ala)₃ through the observation of inter-β-sheet correlations.