FIGURE 6.

Purification and backbone resonance assignments for MMP23-PD. A, MMP23-PD construct used for these studies. Trx, thioredoxin; 6-His, hexahistidine tag; 3C, 3C protease cleavage site; Pro, MMP23 prodomain. B, reverse-phase HPLC purification of MMP23-PD. The monomeric and dimeric forms of MMP23-PD eluted in fractions 4 and 5, respectively. C, SDS-PAGE analysis of purified MMP23-PD. Reverse-phase HPLC fraction numbers are shown in lanes 1 and 2, respectively. D, amino acid sequence of MMP23-PD with the predicted TMD highlighted in red. Spectral overlap prevented assignment of Val-26 (underlined). The sequence includes the N-terminal residual tag residues Gly-1 and Pro-0. E, ¹H-¹⁵N HSQC spectrum of uniformly ¹³C-¹⁵N-labeled MMP23-PD in 20 mm sodium citrate buffer, pH 5.0, containing 100 mm DPC, 20 mm TCEP, 10% ²H₂O, 90% H₂O, and 0.02% w/v NaN₃ at a final protein concentration of 0.7 mm. Inset: Trp indole NH cross-peaks (bottom right) and one of the Gln-52 side chain resonances (center right). Aliased resonances from the nine Arg side chains are highlighted with red boxes.