FIGURE 3.

Oligomeric state analysis of 3CLpro variants. Size-exclusion chromatography profiles of the WT and its variants. Highly concentrated protein samples (180 μM) were injected onto the size-exclusion column (Superdex 200 increase 10/300 GL). The WT exhibited an early large peak corresponding to the dimeric state, with a late smaller peak corresponding to the monomeric state. The chromatographic peaks of His41 variants were different from those of the WT enzyme, with a single dominant peak of dimeric-state protein. Similarly, the chromatographic peak of the C145A variant exhibited a single peak that aligned with the dimeric peak of the WT enzyme. However, the single chromatographic peak of the C145S variant was shifted to an earlier retention volume, which represents a higher oligomeric state than the dimeric state of the WT enzyme.