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. 2020 Dec 17;296:100173. doi: 10.1074/jbc.RA120.016313

Figure 1.

Figure 1

PfA-M17 and Pv-M17 exist as a mix of oligomeric states in solution. FPLC SEC-MALS traces of, A, PfA-M17 and, B, Pv-M17 in 50 mM HEPES pH 8.0, 0.3 M NaCl with approximate sizes calculated using SEC-MALS. Activity of each fraction was measured as an activity rate (fluorescence units/min), normalized, and represented by gray bars. Both PfA-M17 and Pv-M17 are present as a mix of oligomeric states in solution; however, only the large oligomeric state shows substantial catalytic activity. C, c(s) distribution analysis of sedimentation velocity experiments of PfA-M17 (black), Pv-M17 (gray), and PfA-M17 (W525A + Y533A) in 50 mM HEPES pH 8.0, 0.3 M NaCl. Both PfA-M17 and Pv-M17 sedimented as multiple species, whereas PfA-M17 (W525A + Y533A) sedimented as a single monomeric species. Distributions were normalized so that all had a maximum of 100. D, comparative activity levels of PfA-M17 wild type (black) and PfA-M17 (W525A + Y533A) (blue). Enzyme concentrations were uniform between the two samples, and activity levels measured using fluorescence units (FU). PfA-M17 wild type is active, whereas the PfA-M17 (W525A + Y533A) is inactive. FPLC, Fast Protein Liquid Chromatography; SEC-MALS, size exclusion chromatography multiangle light scattering.