Localization of the functional binding domains within Aaa.

<p>Determination of binding of Fg (A, B, C), Fn (D, E, F), and Vn (G, H, I) to the immobilized Aaa (A, D, G), N-Aaa (B, E, H), and C-Aaa (C, F, I) using the BIAcore system. After Aaa, N-Aaa, and C-Aaa were immobilized on the C1 chip surface, Fg, Fn, and Vn were injected over the chip surface at a flow rate of 50 µl/min. I: Binding of Fg, Fn, and Vn was monitored and presented in an overlay-plot of the sensorgrams (a plot of RU [resonance unit] versus time). A, B, C, concentrations of Fg (from bottom to top): 9.16 pM, 18.3 pM, 36.6 pM, 73.2 pM, 146 pM, 293 pM, 586 pM, 1.17 nM, 2.34 nM, 4.69 nM, 9.38 nM, and 18.8 nM. D, E, F, concentrations of Fn (from bottom to top): 293 pM, 586 pM, 1.17 nM, 2.34 nM, 4.69 nM, 9.38 nM, 18.8 nM, 37.5 nM, 75 nM, 150 nM, and 300 nM. G, H, I, concentrations of Vn (from bottom to top): 1.17 nM, 2.34 nM, 4.69 nM, 9.38 nM, 18.8 nM, 37.5 nM, 75 nM, 150 nM, 300 nM, 600 nM, and 1.2 µM. II: Overlay-plot of the sensorgrams (a plot of RU [resonance unit] versus time) of the binding of 4.7 nM Fg to Aaa, N-Aaa, and C-Aaa (A), 18.8 nM Fn to Aaa, N-Aaa, and C-Aaa (B), and 38 nM Vn to Aaa, N-Aaa, and C-Aaa (C) suggesting a 4∶2∶1 stoichiometry for the Fg-binding to Aaa, N-Aaa, and C-Aaa and a 2∶1∶1 stoichiometry for the Vn-binding to Aaa, N-Aaa, and C-Aaa. The stoichiometry for the binding of Fn to Aaa, N-Aaa, and C-Aaa is not completely clear, but appears to be 2∶1∶1.