Signature of Mobile Hydrogen Bonding of Lysine Side Chains from Long-Range ¹⁵N–¹³C Scalar J-Couplings and Computation

Abstract

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 (15)N-(13)C 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 (1)H/(13)C/(15)N heteronuclear correlation experiments selective for lysine NH(3)(+) groups, we analyzed two different types of long-range (15)N-(13)C J-coupling constants: one between intraresidue (15)Nζ and (13)Cγ nuclei ((3)J(NζCγ)) and the other between (15)Nζ and carbonyl (13)C' nuclei across a hydrogen bond ((h3)J(NζC')). The experimental (3)J(NζCγ) data confirm the highly mobile nature of the χ(4) torsion angles of lysine side chains seen in the MD simulation. The NH(3)(+) groups of Lys29 and Lys33 exhibit measurable (h3)J(NζC') couplings arising from hydrogen bonds with backbone carbonyl groups of Glu16 and Thr14, respectively. When interpreted together with the (3)J(NζCγ)-coupling constants and NMR-relaxation-derived S(2) order parameters of the NH(3)(+) groups, they strongly suggest that hydrogen bonds involving NH(3)(+) groups are of a transient and highly dynamic nature, in remarkably good agreement with the MD simulation results.

Time Scales of Slow Motions in Ubiquitin Explored by Heteronuclear Double Resonance

“…The existence of this H-bond has been confirmed by MD simulations 49 and experiments in solution. 50 Alignment of the β-sheets of the two structures ( Figure 4a) offers interesting insight into the conformational transition. The helix α1 is slightly tilted, as if it were pulled on one side by the Glu24-Gly53 H-bond and on the other side by the Lys33-Thr14 Hbond.…”

Effective strategy to assign 1H-15N heteronuclear correlation NMR signals from lysine side-chain NH3 + groups of proteins at low temperature

“…Recently it was demonstrated that lysine (Lys) side-chain NH 3 + groups are very useful probes for NMR studies of protein dynamics involving hydrogen bonds and ion pairs (Anderson et al 2013; Esadze et al 2011; Zandarashvili et al 2013; Zandarashvili et al 2011). Heteronuclear 1 H- 15 N cross peaks from Lys NH 3 + groups are also useful for identifying molecular interfaces between protein and ligand (Blaum et al 2010; Poon et al 2006).…”

“…This 3D experiment relies on relatively small scalar coupling constants 3 J NC between the 15 Nζ and 13 Cγ nuclei. Absolute values of the 3 J NC constants are in a range of 1 – 3 Hz, and depend on Lys χ 4 torsion angles, which can undergo dynamic transitions between gauch and trans conformers (Huang and MacKerell 2013; Zandarashvili et al 2011). Owing to the slow 15 N transverse relaxation of NH 3 + groups during the coherence transfers between the N x and 2 N y C z terms, the magnetizations of interest survive a total of 200 ms for the evolution of the small 3 J NC couplings between the 15 Nζ and 13 Cγ nuclei in the 3D H3NCG experiment.…”

“…In this experiment, undesirable coherence transfer between 15 Nζ and 13 Cε nuclei is avoided by using 13 C I-BURP2 pulses (Geen and Freeman 1991) that invert 13 Cγ (~25 ppm) but do not affect 13 Cε (~42 ppm) nuclei. Although these I-BURP2 pulses also invert 13 Cδ (~28 ppm), 2 J NCδ coupling constants are undetectably small in lysine side chains (Zandarashvili et al 2011). Thus, these 15 N and 13 C shaped pulses permit selective observation of 1 Hζ/ 15 Nζ/ 13 Cγ cross peaks from lysine side chains.…”

“…Our new approaches can help expand applicability of the NMR methods for characterizing Lys side-chain NH 3 + groups (Anderson et al 2013; Andre et al 2007; Esadze et al 2011; Iwahara et al 2007; Segawa et al 2008; Tomlinson et al 2009; Williamson et al 2013; Zandarashvili et al 2013; Zandarashvili et al 2011). As a consequence, dynamics of hydrogen bonds and ion pairs involving Lys side chains may become more assessable for a larger number of proteins and protein complexes.…”