Fig. 3
From: Controlling protein activity by dynamic recruitment on a supramolecular polymer platform
Characterization of BTA-templated enzyme-inhibitor complex formation. a, b Substrate conversion as a function of time by the recruited enzyme in the absence of BLIP (-inhibitor), the fully assembled system (+) and controls lacking one or both of the recruiter strands (-RI, -RE and -RI and -RE, respectively) or the BTA polymers (-BTA). Turnover of a fluorescent substrate (CCF2-FA, 2 μM) was monitored by measuring the fluorescence intensity at 447 nm. Experiments were performed using 25% BTA-DNA (0.5 μM BTA-DNA, 1.5 μM BTA-3OH), 20 nM RE and RI, 1 nM β-lactamase and 10 nM BLIP. Enzyme activities b were obtained from the kinetic traces shown in a by deriving the slope of the initial increase in fluorescence. Enzyme activities were normalized to a control omitting the inhibitor protein. Error bars represent SEM of duplicate measurements. c Normalized enzyme activity as a function of inhibitor concentration (black dots). The enzymatic activities were fitted to Eq. 1, which was derived using the Michaelis–Menten model for competitive inhibition, yielding an apparent inhibition constant (K i,app) of 2.3 ± 0.2 nM (red line). Experiments were performed using 25% BTA-DNA (0.5 μM BTA-DNA, 1.5 μM BTA-3OH), 20 nM RE and 200 nM RI, 1 nM β-lactamase, and 0–100 nM BLIP. Error bars represent s.d. calculated from triplicate measurements. d Normalized enzyme activities as a function of BTA-DNA receptor density. Polymers with receptor densities between 100% and 0.25% BTA-DNA were obtained by assembling a fixed concentration of BTA-DNA (0.5 μM) with varying concentrations of BTA-3OH (0–199.5 μM). Experiments were performed using 20 nM RE and RI, 1 nM β-lactamase and 10 nM BLIP. Error bars represent s.d. calculated from triplicate measurements
