Figure 1.
The molecular structure of avidin family of proteins.
The structure, here represented with biotin-bound avidin (PDB identifier 2AVI), is a homotetramer composed of units of ∼128 residues. Subunits are numbered according to [4]. Each subunit binds one biotin molecule, shown in stick representation. ChiAVD is a hybrid of avidin and AVR4, in which the segment highlighted in orange in avidin (residues 38–60, 23 residues) is replaced by the sequentially related segment found in AVR4 (residues 38–58, 21 residues). Also, Ile 117 of avidin, shown in red, is replaced by a tyrosine found in AVR4.
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].
Figure 3.
Excerpt from the MQ-HNCO-TROSY+ spectrum.
The MQ-HNCO-TROSY+ spectrum of 15N, 13C labeled chimeric avidin was measured at 40°C on 800 MHz in a dilute liquid crystal medium composed of 3∶1 mixture of DMPC:DHPC phospholipids (bicelles). Panel (A) displays overlaid upfield (red contours) and downfield (black contours) components of 15N-{1H} doublets. Vertical dotted lines indicate the position of the corresponding 15N traces shown in panel (B) for T14, N27 and D87. As the scaling parameters were set to κ = 0.5 and λ = 0.5, the measured couplings are scaled down by the factor of 2. The measured apparent 1(J+D)NH/2 couplings are shown next to each splitting for T14, D87 and N27.
Figure 4.
Solution NMR structure of biotin–bound ChiAVD.
(A) Ensemble of fifteen structures of least restraint violations. (B) Lowest–energy structure represented as a ribbon diagram. In each monomer the protein is structured from residues Lys3 to Leu123. β strands in the mean structure span residues 8–12, 17–20, 28–34, 47–53, 63–69, 76–86, 90–100, and 113–122. Residues 106–111 coil to a 310 helix.
Table 1.
Structural Statistics of Biotin–bound ChiAVD.
Figure 5.
Overlay of the NMR structure of biotin–bound ChiAVD and the X–ray structure of biotin–free ChiAVD.
(A) ChiAVD tetramer where the biotin–bound form is represented in dark blue and (B) biotin binding site residues. In (B) aromatic residues are shown in shades of blue (darker colors for the biotin–bound form) and polar residues in shades of green. The 1–2 interface brings Trp110 from the 1–2 related subunit to the binding site.
Figure 6.
Model–free parameters of ChiAVD.
S2, Rex and τe (>0.5 ns) of ChiAVD in biotin–free form at 58°C are shown in black and those of the bound form at 40°C in blue and 58°C in red. The secondary structure of the biotin–bound structure at 58°C is also shown.
Figure 7.
Per residue protection factors of biotin–free and bound ChiAVD.
(A) The protection factors of the free form are shown with red bars. Residues exhibiting fast exchange have been assigned the value 2.5, residues in the second fastest group (see text) the value 2.8, and residues with slow exchange the value 7.0. Residues with no data available have been assigned the value −0.1. Trp side chain data are marked with asterisks. (B) Differences in protection factors shown in the structure of ChiAVD. In dark blue are shown residues with enhanced protection to exchange in both forms and in light blue residues for which the free form is more prone to exchange. Residues shown in red are susceptible to fast exchange in both forms. Residues coloured in white have no comparable data.
Figure 8.
RMSF at three temperatures is shown, in the absence and presence of biotin. The area shaded in grey marks the gap in the amino acid sequence numbering.