Figure 1.

(Top) Sequence homology between wild-type PR1 (without stabilizing mutations) and PR2 (PDB code: 3EBZ) showing identical residues (magenta) and conservative substitutions (light purple). (Bottom left) Structural overlay of PR2 (gold) with autoproteolysis-resistant PR1 (PDB code: 2IEN, shown in cyan). Both crystal structures contain the inhibitor DRV (not shown) in the active site. The left half of the structure shows a surface representation of PR2 with identical residues and conservative substitutions relative to PR1-colored magenta and light purple, respectively. The major site of autoproteolysis, G35/I36, and residue E37 that was mutated to restrict autoproteolysis are indicated by arrows. (Bottom right) Expression of PR2 and its mutants in E. coli and analyses by SDS-PAGE. Lane 1, total soluble extract after initial lysis and centrifugal fractionation. The insoluble pellet obtained from the prior step was briefly sonicated in buffer containing 1M urea and 0.5% NP-40 (see “Materials and Methods” for complete composition) and the supernatant (lane 2) and insoluble (inclusion bodies, lane 3, and duplicate lanes of proteins derived from PR2_E37Q and PR2_E37K mutants) fractions derived after centrifugation are shown. Proteins were subjected to 20% homogeneous PhastGel electrophoresis followed by coomassie staining.