Protein conformational dynamics studied by 15N and 1H R1ρ relaxation dispersion: Application to wild-type and G53A ubiquitin crystals

Abstract

Solid-state NMR spectroscopy can provide site-resolved information about protein dynamics over many time scales. Here we combine protein deuteration, fast magic-angle spinning (~45-60kHz) and proton detection to study dynamics of ubiquitin in microcrystals, and in particular a mutant in a region that undergoes microsecond motions in a β-turn region in the wild-type protein. We use ¹⁵N R_1ρ relaxation measurements as a function of the radio-frequency (RF) field strength, i.e. relaxation dispersion, to probe how the G53A mutation alters these dynamics. We report a population-inversion of conformational states: the conformation that in the wild-type protein is populated only sparsely becomes the predominant state. We furthermore explore the potential to use amide-¹H R_1ρ relaxation to obtain insight into dynamics. We show that while quantitative interpretation of ¹H relaxation remains beyond reach under the experimental conditions, due to coherent contributions to decay, one may extract qualitative information about flexibility.

Keywords: Fast MAS; Protein dynamics; Proton detection; Proton relaxation; Solid-state NMR; Spin relaxation; β-turn.

Figure 1

Conformation of ubiquitin in the βI and βII states. (a) The MPD-ub crystal structure, focusing on the β-turn region and the adjacent α-helix. A neighboring molecule in the crystal is shown in surface representation in grey. The zoom (right insert in (a)) shows a top view of the peptide plane Asp 52/Gly 53 (red ellipse). Note the hydrogen bond to the side chain of Glu 24. This β-turn conformation is called type-II β-turn. (b) Structure of a ubiquitin chain in type-I β-turn conformation (PDB entry 1UBI, yellow) placed inside the crystal arrangement of MPD-ub, by structural alignment. The most striking difference between type-II (a) and type-I (b) structures are the orientation of the peptide plane Asp 52/Gly 53, and the orientation of Glu 24’s side chain. In βI conformation (b) the side chain is rotated outward while it points to Gly 53 in βII. In the context of the crystal packing of MPD-ub, rotating Glu 24 outward would result in a steric clash. This explains why the βI conformation is energetically unfavorable in MPD-ub, whereas in solution and most other crystals βI is favored.

Figure 2

Pulse sequences used in this study for measuring ¹H and ¹⁵N R_1ρ relaxation, using ¹H-to-¹⁵N CP transfers (out and back). A solvent suppression period (“sat.”), using a composite-pulse-decoupling (WALTZ16) proton saturation element while the 15N coherence is stored along +z, is denoted used before the back-transfer to ¹H for detection. ¹⁵N decoupling (WALTZ16) is applied during proton detection with an RF field strength of 3 kHz, and WALTZ16 proton decoupling is used during the indirect ¹⁵N evolution period. Typical ¹⁵N RF field strengths during CP were set to 35 kHz, and the ¹H RF field was chosen to match the n=1 Hartmann-Hahn condition (i.e. ~85 kHz at 50 kHz MAS), with a 90-100% linear ramp.

Figure 3

Assignment of microcrystalline (MPD-ub) G53A ubiquitin. (A) 2D H-N correlation spectrum recorded with a ¹H detected hNH experiment based on cross-polarization steps. (B) Example assignment strips from 3D hCANH, hcoCAcoNH and hcaCBcaNH spectra. The residues shown in (B) are indicated in the 2D spectrum in (A).

Figure 4

G53A in MPD-ub crystals forms predominantly a type-I β-turn. (a) and (b) show psi and phi backbone dihedral angles in WT MPD-ub and G53A MPD-ub, as derived from the assigned chemical shifts, and the program TALOS+[43]. (c) and (d) show the corresponding dihedrals in crystal structures forming a type-I β-turn (here the structure 4XOL[48] was used, which is one out of many βI-forming PDB entries, shown in blue) and type-II β-turn (red, PDB entry 3ONS [32]). The comparison of the psi (residue 52) and phi (residue 53) angle shows that this peptide plane is rotated by the G53A mutation.

Figure 5

¹⁵N R_1ρ relaxation dispersion profiles observed for residues in ubiquitin’s β-turn region and the adjacent helix of G53A MPD-ub. The data were obtained at a MAS frequency of 44.053 kHz. Each data point was obtained from a time series of relaxation data, fitted to a monoexponential function, as described in the Methods section, and the error bars were obtained from Monte Carlo analysis. The dashed lines represent a two-state exchange fit using the Meiboom-1961 model [37], with a common exchange rate constant of 11952 s^-1. The residue-wise ϕ_ex values for these residues are: 0.82 (Asn 25), 0.26 (Ala 53), 0.37 (Thr 55) and 0.24 (Asp 58). In WT MPD-ub the values for residues Ile 23, Val 26, Lys 27, Thr 55 and Asp 58 range between 0.2 and 0.7, i.e. they are of similar magnitude. Relaxation-dispersion data of all other residues are shown in Figure S4.

Figure 6

Numerical simulations of the decay of ¹H magnetization (H_x) in the presence of a spinlock RF field of variable amplitude and stochastic jumps between two states. Shown are the decay rate constants that were obtained from fitting the decay of H_x over the simulated time period (see Methods section for details). In each of the three cases (a-c) the top panel shows that absolute decay rate constant, assuming that the only mechanism present is either the ¹H-¹⁵N dipolar coupling, the ¹H-¹H dipolar coupling or the ¹H CSA tensor, as indicated by different colors. The lower panel shows the relative contributions of these three mechanisms to the total decay rate constant. Note that in each simulation only one relaxation mechanism was present, and that possible cross-correlated relaxation effects are therefore absent. The three panels differ by the assumed exchange rate constant, which was (a) 25·10⁵ s^-1, (b) 1·10⁶ s^-1 and (c) 1·10⁷ s^-1. The motional model is an exchange between the two states depicted in (d); here, the remote proton is at a fixed position, and the NH bond jumps by an angle θ.

Figure 7

Amide proton R_1ρ relaxation data on WT ubiquitin and comparison to ¹⁵N R_1ρ rate constants. Top panel: ¹H R_1ρ obtained at a ¹H Larmor frequency of 950 MHz, using a deuterated sample of MPD-ub in which 70% of the exchangeable sites were reprotonated, and a MAS frequency of 54 kHz. Middle panel: ¹⁵N R_1ρ rate constants reported earlier [45], using a deuterated, 50% reprotonated sample at 600 MHz and 39.5 kHz MAS. Lower panel: correlation of these two data sets; a linear regression curve is shown (slope: 0.15, intercept: -1.35s ^-1, correlation coefficient 0.80).

Figure 8

Amide proton R_1ρ relaxation dispersion data in wild-type ubiquitin microcrystals (MPD-ub), obtained at a MAS frequency of 44.053 kHz and 600 MHz ¹H Larmor frequency. The protein was reprotonated at exchangeable sites at a level of ~35%. The top panel show spin-lock RF-field dependencies for four residues. The lower two panels show ¹H R_1ρ rate constants as a function of residue number obtained at 10 kHz spin-lock RF field strength (top) and the difference of ¹H R_1ρ obtained at RF field strengths of 42 and 10 kHz (bottom). Equivalent data recorded at 800 MHz Larmor frequency and 35 kHz MAS are shown in Figure S5, and reveal very similar behavior, in particular with respect to the first loop and the β-turn region.