Conformational exchange of aromatic side chains by 1H CPMG relaxation dispersion

Ulrich Weininger,Heiner N. Raum,Matthias Dreydoppel

doi:10.1007/s10858-018-0210-5

Ulrich Weininger, Heiner N. Raum + Show 1 more

Open Access

https://doi.org/10.1007/s10858-018-0210-5

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Abstract

Aromatic side chains are attractive probes of protein dynamics on the millisecond time scale, because they are often key residues in enzyme active sites and protein binding sites. Further they allow to study specific processes, like histidine tautomerization and ring flips. Till now such processes have been studied by aromatic 13C CPMG relaxation dispersion experiments. Here we investigate the possibility of aromatic 1H CPMG relaxation dispersion experiments as a complementary method. Artifact-free dispersions are possible on uniformly 1H and 13C labeled samples for histidine δ2 and ε1, as well as for tryptophan δ1. The method has been validated by measuring fast folding–unfolding kinetics of the small protein CspB under native conditions. The determined rate constants and populations agree well with previous results from 13C CPMG relaxation dispersion experiments. The CPMG-derived chemical shift differences between the folded and unfolded states are in good agreement with those obtained directly from the spectra. In contrast, the 1H relaxation dispersion profiles in phenylalanine, tyrosine and the six-ring moiety of tryptophan, display anomalous behavior caused by 3J 1H–1H couplings and, if present, strong 13C–13C couplings. Therefore they require site-selective 1H/2H and, in case of strong couplings, 13C/12C labeling. In summary, aromatic 1H CPMG relaxation dispersion experiments work on certain positions (His δ2, His ε1 and Trp δ1) in uniformly labeled samples, while other positions require site-selective isotope labeling.Graphical abstract

Highlights

Proteins are dynamic entities that continuously undergo dynamic processes on various time scales
Conformational transitions on the millisecond time scale are often linked to biological function (Mittermaier and Kay 2009) and transiently populated high-energy states play important roles in enzyme catalysis (Boehr et al 2006; Cole and Loria 2002; Eisenmesser et al 2002) or ligand binding (Demers and Mittermaier 2009; Malmendal et al 1999)
Such transitions between different conformations generally lead to a modulation of NMR parameters as the chemical shift (Gutowsky and Saika 1953) or residual dipolar couplings (Igumenova et al 2007; Vallurupalli et al 2007), resulting in exchange contributions to transverse relaxation rate constants

Summary

Introduction

Proteins are dynamic entities that continuously undergo dynamic processes on various time scales. Conformational transitions on the millisecond time scale are often linked to biological function (Mittermaier and Kay 2009) and transiently populated high-energy states play important roles in enzyme catalysis (Boehr et al 2006; Cole and Loria 2002; Eisenmesser et al 2002) or ligand binding (Demers and Mittermaier 2009; Malmendal et al 1999). Such transitions between different conformations generally lead to a modulation of NMR parameters as the chemical shift (Gutowsky and Saika 1953) or residual dipolar couplings (Igumenova et al 2007; Vallurupalli et al 2007), resulting in exchange contributions to transverse relaxation rate constants. Residual dipolar couplings have been obtained (Sathyamoorthy et al 2013)

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