Figure 1. Effects of cassette sequence on GFP unfolding and degradation.

(A) Starting at the N terminus, substrates contained residues 1–229 of A.victoria GFP (PDB 1GFL, Yang et al., 1996), a cassette with 12 variable residues, and a partial ssrA degron. (B) Method for measuring intracellular degradation of substrates by ClpX^ΔN/ClpP. (C) Cellular fluorescence depends upon ClpX^ΔN/ClpP expression and cassette sequence (listed in Table 1). (D) Fraction intracellular degradation for substrates bearing different cassettes. (E) Fits of the substrate dependence of degradation in vitro to a hyperbolic Michaelis-Menten equation. (F) V_max values for different substrates. In panels, C–F, values represent averages (± S.D.) of three biological replicates.