Fig. 2.

Degradation activity and properties of mf-Lon. (A) SDS/PAGE assays of degradation of carboxylmethylated titin-I27 (5 μM) with different tags by mf-Lon or ec-Lon (150 nM hexamer). (B) Degradation of fluorescein-labeled titin-I27-mf-ssrA (5 μM) by mf-Lon (150 nM hexamer) required ATP and was not observed with ADP, without nucleotide, or in the absence of enzyme. Proteolysis resulted in an increase in fluorescence. (C) Rates at different temperatures for degradation of carboxylmethylated ³⁵S-labeled titin-I27-mf-ssrA (3 μM) by mf-Lon (30 nM hexamer) were determined at pH 7.5 by release of acid-soluble peptides. (D) Rates at different pH values for degradation of carboxylmethylated ³⁵S-labeled titin-I27-mf-ssrA (3 μM) by mf-Lon (30 nM hexamer) were determined at 30°C. The maximal degradation rate is higher than in C because the sodium phosphate buffer (50 mM) used in the temperature experiments was slightly inhibitory. (E) Michaelis–Menten plots. Steady-state rates of degradation of an unfolded substrate (carboxylmethylated ³⁵S-titin-mf-ssrA) or a native substrate (³⁵S-titin-mf-ssrA) by mf-Lon (10 nM hexamer) were assayed by release of acid-soluble peptides and plotted as a function of substrate concentration. The solid curves are fits (R² ≥ 0.99) to the Hill form of the Michaelis–Menten equation (V = V_max·[S]ⁿ/(K_mⁿ + [S]ⁿ). (F) The titin-I27 protein (4 μM; filled circles) and titin-I27-mf-ssrA protein (4 μM; open circles) had the same thermal stability, as assayed by changes in CD ellipticity at 228 nm. Ellipticity data were fitted to a two-state model by nonlinear regression (46). (Inset) CD spectra at 25°C show that both titin variants (40 μM) are natively folded. The differences in the two spectra result from the contribution of the long unstructured mf-ssrA tag to the CD signal.