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

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.

Keywords: Aromatic side chains; Conformational exchange; Protein dynamics; Strong couplings.

Fig. 1

Aromatic side chains (His, Trp, Phe and Tyr) with different positions labeled. δ1, δ2 and ε1, ε2 in Phe and Tyr are usually averaged to δ* and ε* because of fast ring flips. His is shown in its most stable neutral tautomeric form

Fig. 2

Pulse sequence of the ¹H CPMG relaxation dispersion experiment for measuring conformational exchange of aromatic side chains. All pulses are applied along the x-axis unless otherwise indicated. Narrow (wide) solid bars indicate rectangular high-power 90° (180°) pulses. Wide grey bars indicate 180° pulses in the CPMG elements, which have attenuated power. The wide semi-ellipse on ¹³C represents a REBURP (Geen and Freeman 1991) pulse with a bandwidth of 40 ppm. All proton pulses after (a) are applied on resonant on the aromatics, after (b) on-resonant on water. The delay τ can be set to 1.6 ms (Phe and Tyr), 1.35 ms (all aromatics) or 1.25 ms (His). T_a and T_b are 1.1 ms and 1.1 ms + t₁/2 for non constant-time detection and 4.464 ms − t₁/4 and 4.464 ms + t₁/4 for constant-time detection, respectively. The pulses flanking the CPMG blocks purge non-refocused magnetization remaining as a consequence of the variation among aromatic sites in the ¹J_HC coupling constant (Vallurupalli et al. ; Weininger et al. 2012c). The phase cycle is: ϕ₁ = (x, − x), ϕ₂ = (x, x, − x, − x), ϕ_rec = (x, − x, − x, x). Pulsed field gradients G1–3 are employed to suppress unwanted coherences and artifacts, while GC and GH are encoding and decoding gradients, respectively, for echo/anti-echo coherence selection, obtained by inverting the signs of GH (Kay et al. 1992). For every second t₁ increment ϕ₁ and the receiver were incremented. Gradient durations (in ms) and relative power levels (in %) are set to (duration, power level) G1 = (1.0, 13), G2 = (0.5, 10), G3 = (1.0, 90), GC = (1.0, 80), GH = (1.0, − 20.1)

Fig. 3

Simulation of additional R₂ contributions in aromatic ¹H CPMG relaxation dispersion profiles caused by ³J ¹H–¹H couplings. Simulations were performed with Qsim (Helgstrand and Allard 2004). a Assuming a J-coupling of 2 Hz, which is the maximal J-coupling in case of His δ2, His ε1 and Trp δ1 (RMSD 0.12 s⁻¹). b Assuming a J-coupling of 8 Hz, which is the coupling in Phe, Tyr and the six-ring moiety of Trp. Here, simulations were performed for one coupling ¹H (black circles), two coupling ¹H (black squares) or one coupling ¹H which itself is directly coupling to ¹³C (red circles)

Fig. 4

Aromatic ¹H CPMG relaxation dispersion profiles of ubiquitin H68 δ2 (a), H68 ε1 (b) and GB1 W43 δ1 (c). Black symbols represent a close to on-resonant carrier during the CPMG block, red symbols an off-resonant carrier. RMSD values for on-resonant experiments (black symbols) are 0.18 s⁻¹, 0.12 s⁻¹, 0.26 s⁻¹

Fig. 5

Aromatic ¹H CPMG relaxation dispersion profiles of ubiquitin (a–c) F4 δ* (a), F4 ε* (b), Y59 δ* (c), tSlyD (d–f) F79 δ* (d), F91 δ* (e), F117 δ* (f), GB1 (g–l) W43 ε3 (g), W43 ζ3 (h), W43 η2 (i), F52 δ* (j), Y33 δ* (k) and Y33 ε* (l). Red symbols represent measurements on uniformly ¹³C labeled samples, black symbols measurements on site-selective ¹³C labeled samples

Fig. 6

Ribbon representation of CspB using 1csp.pdb. The ribbon is colored in blue, side chains of W8 and are shown as sticks, labeled and colored in red. The figure was generated using PyMOL (Schrodinger 2010)

Fig. 7

Aromatic ¹H CPMG relaxation dispersion profiles acquired on a 1.8 mM sample of CspB in 10 mM HEPES pH 7.0 at 25 °C and static magnetic field strengths of 14.1 T (blue) and 18.8 T (red). Data are shown for H29 δ2 (a), H29 ε1 (b) and W8 δ1 (c). Solid lines represent the global fit to the experimental data

Fig. 8

Correlation of ¹H chemical shift differences between the folded and unfolded states of CspB derived from CPMG relaxation dispersion experiments under native conditions and measured directly from ¹H–¹³C HSQC spectra in an urea titration experiment. The solid black line represents the ideal correlation