In vitro purified STRAP binds JMY and inhibits both intrinsic nucleation activity and the ability to activate the Arp2/3 complex. (A) FRET assay for the binding of purified STRAP to JMY. The curves are fluorescence intensity as a function of emission wavelength of 160 nM Alexa Fluor 546–labeled JMY (green) as donor, 600 nM Alexa Fluor 647–labeled STRAP (pink) as acceptor, and mixture of both fluorescently labeled proteins (red). The traces of donor alone and acceptor alone are combined to serve as baseline (blue), indicating no interaction. FRET is detected as an increase in acceptor emission intensity compared with baseline. (B) Binding isotherm for STRAP-JMY interaction derived from FRET assay (Kd = ∼300 nM). Alexa Fluor 546–labeled JMY is titrated in 300 nM Alexa Fluor 647–labeled STRAP in 20 mM Hepes buffer, pH 7.4, 100 mM KCl. (C) STRAP inhibits JMY’s intrinsic nucleation activity. Actin assembly was monitored by pyrene-actin fluorescence in various concentrations of STRAP (left). The initial slope (first 250 s) of each curve was normalized and plotted as a proxy for nucleation rate (right). (D) Dose dependence of STRAP inhibition of Arp2/3 complex activation by JMY. Pyrene-actin polymerization assay was performed in 50 mM KCl, 1 mM MgCl2, 1 mM EGTA, and 10 mM Imidazole, pH 7.0, buffer with 1 µM (5% labeled) actin, 200 nM JMY, and 25 nM Arp2/3 as noted and STRAP as indicated.
