Chimeric Avidin – NMR Structure and Dynamics of a 56 kDa Homotetrameric Thermostable Protein
Figure 2
MQ-HNCO-TROSY+ experiment for the measurement of 15N-1H residual dipolar couplings in 15N, 13C, (2H) labeled proteins.
(A) Narrow and wide black bars denote rf pulses with 90° and 180° flip angles, respectively. If not otherwise denoted, the pulses are applied with phase x. The 1H, 15N, 13C′, and 13Cα carrier positions are 4.7 (water), 118 (center of 15N spectral region), 175 ppm (center of 13C′ spectral region), and 57 ppm (center of 13Cα spectral region). 90° (180°) pulses for 13C′ are applied with a strength of Ω/√15 (Ω/√3), where Ω is the frequency difference between the centers of the 13C′ and the aliphatic 13Cα regions. The 13C carrier is placed in the middle of 13C′ region (175 ppm) and rectangular 180° pulses are applied off-resonance for 13Cα with phase modulation by Ω. Removal of 13C′–13Cα and 15N–13Cα coupling interactions during t1 and t2, respectively, can be accomplished using either the SEDUCE-1 decoupling sequence [37] or three 180° 13Cα rectangular pulses applied off-resonance with phase modulation by Ω. The delays used for coherence transfer are: Δ = 1/(4JNH); TN = 1/(4JNC′) = 12.5–16.6 ms; ε = duration of gradient + recovery delay. Inset (A′) shows implementation to select the anti-TROSY component, which is downscaled by a factor of κ (0<κ<1) with respect to the TROSY component (see panel B). The phase cycling used is: φ1 = x, −x; φ2 = x; φ3 = −x; φ4 = −x; ψ = −x; φrec. = x, −x. Inset (A") shows pulse sequence implementation to select the anti-TROSY component which is scaled up by a factor of λ (λ>0) with respect to the TROSY component (see panel C). The phase cycling used is: φ1 = x, −x; φ2 = x; φ3 = −x; φ4 = −x; ψ = x; φrec. = x, −x. Hence, for measuring 1JNH (and 1(J+D)NH) couplings, the κ and λ values can be selected independently, for instance using κ = 0; λ = 1 yields two subspectra whose resonance frequencies differ by 2πJNH i.e. 1JNH couplings can be obtained directly from the frequency separation. Quadrature detection in the indirect 15N (t2) dimension, the 90°(15N) with the phase ψ is inverted simultaneously with the gradient GN to obtain echo/antiecho selection. The data processing is according to the sensitivity enhanced method [38]. The axial peaks are shifted to the edge of the spectrum by inverting φ2 together with φrec. in every second t2 increment. Quadrature detection in the 13C′ dimension is obtained by States-TPPI protocol applied to φ1 [39].