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. 2015 Jan 2;290(8):4728–4740. doi: 10.1074/jbc.M114.605956

FIGURE 1.

FIGURE 1.

Effect of the Kly18 propeptide in vitro and in vivo. A, scheme depicting the domain structure of T. forsythia karilysin. Numbering according to UniProt D0EM77. B, SDS-PAGE of cultures of wild-type and E156A variants of pKly18 and Kly18. Lanes 1 and 2, insoluble and soluble fractions of wild-type pKly18 (from pKAR10), respectively. Lanes 3 and 4, insoluble and soluble fractions of wild-type Kly18 (pKAR12), respectively. Lanes 5 and 6, insoluble and soluble fractions of pKly18-E156A (pKAR11), respectively. Lanes 7 and 8, insoluble and soluble fractions of Kly18-E156A (pKAR13), respectively. Overexpressed proteins are labeled with an asterisk. C, unfolding transition curves showing temperature-dependent change in fluorescence of pKly18-E156A (pKAR5; solid line) and Kly18-E156A (pKAR6; dashed line) in the absence (black curve) and presence of CaCl2 (1 mm, red curve; 5 mm, green curve). The blank curve is indicated with a dotted line. D, proteolytic activity of Kly18 (pKAR1) at 37 °C using substrate Mca-R-P-K-P-V-E-Nva-W-R-K(dnp)-NH2 at 10 μm in the absence (0) and presence of 0.1, 0.5, 1, 2, and 5 mm propeptide mimic. E and F, stability of mutant pKly-Y35A (E) and mutant pKly-Y35A/D25A (F) over time. Kly48, Kly38, and Kly18 are indicated by arrows.