NMR analysis of the MIP-MDM2 fusion and MIP:MDM2 complex.

<p>(<b>A</b>) Schematic diagram of the HAT-GB1 fused MIP-MDM2-T7tag protein expression plasmid. The T7 promoter, ribosome binding site (RBS), restriction enzyme sites, and protease cleavage sites are also indicated. (<b>B, left</b>) Shown in orange is the 2D ¹H-¹⁵N HSQC spectrum of the MIP-MDM2-T7tag linked protein (MIP-MDM2) and in cyan the MIP:MDM2-T7tag complex (MIP:MDM2 complex). (<b>B, right</b>) 2D ¹H-¹⁵N HSQC spectrum of MIP-MDM2. Signals are labeled with the residue number and a one-letter amino acid code: The MIP, TEV cleavage site, and MDM2-T7tag portions are colored brown, gray, and blue, respectively. (<b>C, left</b>) Chemical shift differences of the corresponding signals in Fig. 1 (B, left). The chemical shift difference, Δδ, was determined as Δδ = [(Δδ_H)²+ (Δδ_N/6.5)²]^1/2, where Δδ_H and Δδ_N are the chemical shift differences for HN and ¹⁵N, respectively. The mean value and the mean value +1SD are shown by solid and dashed lines, respectively. (<b>C, right</b>) Steady-state ¹H-¹⁵N NOE values are shown for MIP-MDM2. “P”s indicate proline residues and asterisks indicate residues whose ¹H-¹⁵N resonance was not assigned.