Signature of mobile hydrogen bonding of lysine side chains from long-range ¹⁵N-¹³C scalar J-couplings and computation

Amino acid side chains involved in hydrogen bonds and electrostatic interactions are crucial for protein function. However, detailed investigations of such side chains in solution are rare. Here, through the combination of long-range ¹⁵N-¹³C scalar J-coupling measurements and an atomic-detail molecular dynamics (MD) simulation, direct insight into the structural dynamic behavior of lysine side chains in human ubiquitin has been gained. On the basis of ¹H/¹³C/¹⁵N heteronuclear correlation experiments selective for lysine NH₃ ⁺ groups, we analyzed two different types of long-range ¹⁵N-¹³C J-coupling constants: one between intraresidue ¹⁵Nζ and ¹³CCγ nuclei (³J _NζCCγ) and the other between ¹⁵Nζ and carbonyl ¹³C′ nuclei across a hydrogen bond ( ^h3J_NζC′). The experimental ³J _NζCCγ data confirm the highly mobile nature of the Ξ₄ torsion angles of lysine side chains seen in the MD simulation. The NH₃⁺ groups of Lys29 and Lys33 exhibit measurable ^h3J_NζC′ couplings arising from hydrogen bonds with backbone carbonyl groups of Glu16 and Thr14, respectively. When interpreted together with the ³J_NζCCγ-coupling constants and NMR-relaxation-derived S² order parameters of the NH ₃⁺ groups, they strongly suggest that hydrogen bonds involving NH₃⁺ groups are of a transient and highly dynamic nature, in remarkably good agreement with the MD simulation results.