Protein backbone motions viewed by intraresidue and sequential HN-Halpha residual dipolar couplings

Abstract

Triple resonance E.COSY-based techniques were used to measure intra-residue and sequential H(N)-H(alpha) residual dipolar couplings (RDCs) for the third IgG-binding domain of protein G (GB3), aligned in Pf1 medium. Measurements closely correlate with values predicted on the basis of an NMR structure, previously determined on the basis of a large number of one-bond backbone RDCs measured in five alignment media. However, in particular the sequential H(N)-H(alpha) RDCs are smaller than predicted for a static structure, suggesting a degree of motion for these internuclear vectors that exceeds that of the backbone amide N-H vectors. Of all experimentally determined GB3 structures available, the best correlation between experimental (1)H-(1)H couplings is observed for a GB3 ensemble, previously derived to generate a realistic picture of the conformational space sampled by GB3 (Clore and Schwieters, J Mol Biol 355:879-886, 2006). However, for both NMR and X-ray-derived structures the (1)H-(1)H couplings are found to be systematically smaller than expected on the basis of alignment tensors derived from (15)N-(1)H amide RDCs, assuming librationally corrected N-H bond lengths of 1.041 A.

Figure 1

Pulse sequence of the 3D HN(CO)CA[HA]-E.COSY experiment for measurement of sequential D_HαHN couplings. The radio-frequency pulses on ¹H, ¹⁵N, ¹³C^α and ¹³C’ are applied at 4.7, 120, 56 and 174 ppm, respectively. Narrow and wide bars indicate non-selective 90° and 180° pulses. The triangular shape represents a ¹³C’-selective 180°-Gaussian pulse of duration p₁ = 150 μs; the shaped ¹H^N-selective EBURP and ReBURP pulses (Geen and Freeman, 1991) have durations p₂ = 1.0 ms and p₃ = 1.5 ms and are applied at 10.2 ppm and 9.9 ppm, respectively, on a 600 MHz instrument. The duration of the shaped pulses needs to be adjusted inversely with the strength of the static magnetic field. ¹⁵N-decoupling is achieved with WALTZ16 (Shaka et al., 1983) at a RF field strength γB₁ = 1.1 kHz, and optional ¹³C’-decoupling is also achieved with WALTZ16 (γB₁ = 600 Hz). Unless indicated otherwise, all radio-frequency pulses are applied with phase x. The phase cycle is: ϕ₁ = x; ϕ₂ = {x, y,-x,-y}; ϕ₃ = x; ϕ₄ = {x, x,-x,-x}; ϕ₅ = {x, x, x, x,-x,-x,-x,-x}; ϕ_rec = {x,-x,-x, x,-x, x, x,-x}. The delays have the following values: τ₁ = 1/(4J_HN) = 2.6 ms, τ₂ = 1/(4J_NC’) = 13.5 ms, τ₃ = 6.5 ms = 0.7 × 1/(2J_CαC’), τ₄ = 11 ms < 1/(4J_NC) and η is the length of the ¹³C^α 180° pulse. Pulsed field gradients (PFG) are applied along the z-axis with duration/strength of: G₁, 0.5 ms / 12 G/cm; G₂, 2 ms / 18 G/cm; G₃, 1.5 ms / -10 G/cm; G₄, 0.7 ms / -18 G/cm; G₅, 1 ms / 15 G/cm; G₆, 0.2 ms / 18 G/cm. Quadrature detection in the ¹⁵N (t₁) and ¹³C(t₂) dimension is achieved by the States-TPPI method, applied to the phases ϕ₁ and ϕ₃, respectively.

Figure 2

Small regions of (F₂,F₃) cross sections through the HNCA[HA] (top, intraresidue) and HN(CO)CA[HA] (bottom, sequential) E.COSY spectra of GB3 taken at the ¹⁵N frequencies of Asn8 (left) and Trp43 (right). Multiplets exhibit the ¹J_CαHα + ¹D_CαHα splitting in the ¹³C dimension, and the J_HαHN + D_HNHα displacement in the H^N dimension.

Figure 3

Correction in Hz to be applied to the (D + J) value as measured from the E.COSY spectra, as a function of (D + J) and R₁^Hα. Opposite sign corrections of the same magnitude are required for interactions where (D + J) < 0. Corrections were derived using peak picking on multiplets simulated using eq 9, assuming H^N Lorentzian (left) or Gaussian (right) line shapes with line width at half height of 35 Hz, and H^α spin flips occurring between the end of the C^α evolution period and the start of direct detection (28.8 ms).

Figure 4

Correlation plots showing predicted versus experimental RDCs for (a) ³D_HNHα and (b)⁴D_HαHN for 2OED. The effective alignment tensor is obtained from the ¹D_HN RDCs and used for the prediction of all RDCs (r_NH^eff is 1.041 Ǻ). Table 1 presents statistics regarding the observed correlations. Highly flexible residues 12, 40 and 41 are excluded. Symbols embossed with red asterisks correspond to residues that fall more than one standard deviation from a linear regression best fit. Residue labeling corresponds to the residue on which H^N resides.

Figure 5

Correlation plots showing predicted versus experimental RDCs for (a) ³D_HNHα and (b) ⁴D_HαHN, for a 160 conformer ensemble (Clore and Schwieters, 2006). The effective alignment tensor is obtained from the ¹D_HN RDCs and used for the prediction of all other RDCs (r_NH^eff is 1.041 Ǻ). Table 1 presents statistics regarding the observed correlations. Symbols embossed with red asterisks correspond to RDCs that differ by more than 1.5 standard deviations from a linear regression best fit. Residue labeling for ⁴D_HαHN corresponds to the residue on which H^N resides.

Figure 6

RMSD between experimental and predicted sequential D_HNHα couplings in GB3 versus r_HN and r_CαHα bond lengths for the 160-conformer ensemble. Equidistant lines are drawn in steps of 0.03 Hz above the minimal rmsd of 1.16 Hz (at r_HN = 1.05 Ǻ and r_CαHα = 1.09 Ǻ). The blue ellipse covers bond length pairs with an rmsd which is not statistically significantly larger than the minimal rmsd (<1.19 Hz).