Revealing weak histidine ¹⁵N homonuclear scalar couplings using Solid-State Magic-Angle-Spinning NMR spectroscopy

The tautomeric structure and chemistry of the histidine imidazole ring play active roles in many structurally and functionally important proteins and polypeptides. While in NMR spectroscopy histidine chemical shifts (e.g. ¹⁵N, ¹³C, and ¹H) have been commonly used to characterize the tautomeric structure, hydrogen bonding, and torsion angles, homonuclear ¹⁵N scalar couplings in histidine have rarely been reported. Here, we propose double spin-echo sequences to compare the observed signals with and without a 90° pulse between the two spin-echo periods, such that their signal ratio as a function of the echo time solely depends on homonuclear scalar couplings, allowing for measuring weak homonuclear scalar couplings without influence from transverse dephasing effects, thus capable of revealing hydrogen-bond mediated ¹⁵N-¹⁵N J-couplings that can provide direct and definitive evidence for the formation of N^…H^…N hydrogen-bonding associated with the imidazole ring. We used two ¹³C,¹⁵N labeled histidine samples recrystallized from solutions at pH 6.3 and pH 11.0 to demonstrate the feasibility of this method and reveal the existence of a weak two-bond scalar coupling between the N_δ1 and N_ε2 sites in the histidine imidazole ring in three tautomeric states and the presence of a hydrogen-bond mediated scalar coupling between the N_δ1 site in the imidazole ring and the backbone N_α site in the histidine neutral τ and π states. Our results demonstrate that weak ¹⁵N homonuclear scalar couplings can be measured even when their values are less than their corresponding intrinsic natural linewidths, thus providing direct and definitive evidence for the formation of N^…H^…N hydrogen bonding that is associated with the histidine imidazole ring.