Five and four dimensional experiments for robust backbone resonance assignment of large intrinsically disordered proteins: application to Tau3x protein

Abstract

New experiments dedicated for large IDPs backbone resonance assignment are presented. The most distinctive feature of all described techniques is the employment of MOCCA-XY16 mixing sequences to obtain effective magnetization transfers between carbonyl carbon backbone nuclei. The proposed 4 and 5 dimensional experiments provide a high dispersion of obtained signals making them suitable for use in the case of large IDPs (application to 354 a. a. residues of Tau protein 3x isoform is presented) as well as provide both forward and backward connectivities. What is more, connecting short chains interrupted with proline residues is also possible. All the experiments employ non-uniform sampling.

Keywords: High-dimensionality NMR; Intrinsically disordered proteins; Isotropic mixing; MOCCA-XY16; Non-uniform sampling; Resonance assignment.

Fig. 1

Coherence pathway schemes for 5D (H)NCOCONH (top) and 5D (HACA)CON(CO)CONH (bottom) experiments

Fig. 2

(H)NCOCONH (top) and (HACA)CON(CO)CONH (bottom) experiments pulse sequence schemes. Rectangles represent hard pulses. Filled and empty symbols represent 90° and 180° pulses, respectively. ¹H and ¹⁵N composite pulse decoupling is performed with WALTZ-16 (Shaka et al. 1983), at γB₁/2π of 5.1 and 1.14 kHz at 800 MHz spectrometer, and 3.37 and 0.97 kHz at 600 MHz, respectively. Simultaneous inversion of CA and CO spins is achieved using a 6-element composite pulse (Shaka 1985). Selective CA and CO ¹³C pulses are applied with the RF field strength adjusted to |ΔΩ_CA − CO|/√15 (√3) for 90° and 180° pulses, respectively. For the 800 MHz spectrometer 90° and 180°, rectangular and sinc-shaped pulsed (bell-shaped at the scheme) with a duration of 40.1 (35.9) µs, and 65.8 (58.8) µs, respectively, are used. Whereas, for the 600 MHz spectrometer the respective pulse lengths are 53.5 (47.9) µs and 87.8 (78.4) µs. Off-resonance pulses are applied using phase modulation of the carrier. The PFG durations are set to 0.5 ms, except for coherence selection gradients for which 2.0 and 0.2 ms, are used. Delay durations are set as follows: ΔNH = 5.4 ms, ΔNCO = 28 ms, ΔCOCA = 9.1 ms. Evolution periods for H, N, CO are in semi-constant-time mode: a_i = (t_i + Δ)/2; b_i = t_i(1 − Δ/t_imax)/2; c_i = Δ(1 − t_i/t_imax)/2 or in constant-time mode: a_i = (t_i + Δ)/2; b_i = 0; c_i = (Δ − t_i)/2 where Δ stands for ΔNH, ΔNCO. In the second sequence evolution for N in t₂ is in real-time mode. The four step phase cycle is: ϕ₁ = x, −x; ϕ₂ = 2x, 2(−x) and ϕ_rec = x, 2(−x), x. In t₁, t₂, t₃, t₄ dimensions quadrature is accomplished using States-TPPI method, by incrementing ϕ₁, ϕ₂, ϕ₃, ϕ₄ phases, respectively. The phase ψ = x is inverted simultaneously with the last gradient pulse to achieve echo-antiecho coherence transfer selection in the last indirect dimension. The coherence selection gradients marked with xyz are applied at the magic angle (600 MHz) or along the z axis (800 MHz). 180° water 1.42 (1.07) ms sinc-shaped flipback pulses are used, on the 600 and 800 MHz spectrometers, respectively

Fig. 3

Resulting 2D cross-sections from 5D (H)NCOCONH (top) and 5D (HACA)CON(C)CONH for residues 105–111 of α-synuclein (strip plot for P108 is not shown). Diagonal peak positions are marked with black crosses. Note the presence of cross-peaks A107CO-P108N on 3rd and 4th panels (bottom), which allow sequential assignment despite the presence of a proline residue

Fig. 4

Tau3x protein sequence with resonance assignment of CON pairs obtained using: 3D HNCO, 5D HN(CA)CONH, 5D (HACA)CON(CA)CONH, 5Dn HabCabCONH and 5D HC(CC-TOCSY)CONH. Green CON pair assigned, black only CO assigned, blue only N assigned, red no assignment

Fig. 5

Tau3x CON projection taken from a 3D HNCO spectrum (left). Zoom into the most crowded part of the CON projection (right)

Fig. 6

Strip plots for the residues from A101 to G151 taken from 4D (HACA)CO(NCO)CONH of Tau3x. Diagonal peak positions are marked with black crosses. Strips plots for proline residues are not shown. ¹³C chemical shifts of residues preceding prolines are evaluated from cross-peaks present on neighbouring residues’ strip plots

Fig. 7

Strip plots for the residues from A101 to G151 taken from 4D (HACACO)N(CO)CONH of Tau3x. Diagonal peak positions are marked with black crosses. Strips plots for proline residues are not shown, nonetheless their ¹⁵N chemical shifts are evaluated from cross-peaks present on neighbouring residues’ strip plots. The small, lower ¹⁵N frequency peaks originate from ²H¹⁵N–¹H¹⁵N isotopomers

Fig. 8

Tau3x protein sequence with resonance assignment of CON pairs obtained using 3D HNCO, 4D (HACA)CO(NCO)CONH and 4D (HACACO)N(CO)CONH. Green CON pair assigned, black only CO assigned, blue only N assigned. Red background indicates correction of a previous assignment