Site-selective 1H/2H labeling enables artifact-free 1H CPMG relaxation dispersion experiments in aromatic side chains

Abstract

Aromatic side chains are often key residues in enzyme active sites and protein binding sites, making them attractive probes of protein dynamics on the millisecond timescale. Such dynamic processes can be studied by aromatic 13C or 1H CPMG relaxation dispersion experiments. Aromatic 1H CPMG relaxation dispersion experiments in phenylalanine, tyrosine and the six-ring moiety of tryptophan, however, are affected by 3J 1H–1H couplings which are causing anomalous relaxation dispersion profiles. Here we show that this problem can be addressed by site-selective 1H/2H labeling of the aromatic side chains and that artifact-free relaxation dispersion profiles can be acquired. The method has been further validated by measuring folding–unfolding kinetics of the small protein GB1. The determined rate constants and populations agree well with previous results from 13C CPMG relaxation dispersion experiments. Furthermore, the CPMG-derived chemical shift differences between the folded and unfolded states are in excellent agreement with those obtained directly from the spectra. In summary, site-selective 1H/2H labeling enables artifact-free aromatic 1H CPMG relaxation dispersion experiments in phenylalanine and the six-ring moiety of tryptophan, thereby extending the available methods for studying millisecond dynamics in aromatic protein side chains.

1H R1ρ relaxation dispersion experiments in aromatic side chains

“…Y33ε, F52ε, and W43ζ3 (Figs. 3b, d, f) show flat dispersion profiles in the deuterated sample, again in agreement with expectations (very fast exchange, no 1 H chemical shift difference between the symmetric sites, and no exchange, respectively; see above). In the case of F30ε, the site-selectively deuterated sample reveals a fast exchange process that is completely hidden by the strong-coupling effect in the protonated sample (Fig.…”

“…3c, d) compared to Tyr (Fig. 3a, b), because the investigated 1 Hε spins in Phe couple to two vicinal proton spins (δ and ζ) instead of one (δ) in Tyr. In the case of Trp ζ3, the magnitude is closer to the Tyr case.…”

“…All experiments were acquired on a Bruker Avance III spectrometer at a static magnetic field strength of 14.1 T, equipped with a room temperature probe. 1 H R 1ρ relaxation dispersion experiments were performed using the pulse sequence shown in Fig. 2, with spectral widths of 14.0 ppm ( 1 H) and 30.0 ppm ( 13 C), by 1024 and 128 points, respectively.…”

“…3, together with predicted apparent R 1ρ dispersion profiles (black lines), calculated using Eq. 6 and the artifact-free data measured on deuterated samples (blue points) 1 H R 1ρ relaxation dispersion experiments acquired on uniformly protonated samples.…”

Transition-State Compressibility and Activation Volume of Transient Protein Conformational Fluctuations

“…5 − 18 In fact, experimental characterization of ring flip rates did not progress much after the groundbreaking observations in the 1970s, partly because of limitations of the NMR technique, which required well-resolved signals in the slow-exchange regime. Recently, there has been a renaissance in these types of studies, 11 , 13 , 19 , 20 enabled by methodological advances, including site-selective isotope labeling of aromatic side chains, 21 − 27 relaxation dispersion experiments tailored to aromatic rings, 28 − 31 and improved analysis that takes into account the effects of strong 1 H– 1 H J -coupling within the aromatic ring. 16 …”