Figure 2.

rFHR-1 and rFHR-2 form homo- and heterodimers in vitro, while rFHR-5 forms homodimers only. (A) Schematic overview of a monomer exchange experiment, to monitor reaction kinetics between fluorescently labeled recombinant human FHRs (rFHRs). Excitation of the 488-labeled monomer will lead to FRET toward the 594-labeled monomer, when in close proximity (B) rFHR-2⁴⁸⁸ and rFHR-2⁵⁹⁴ or (C) rFHR-5⁴⁸⁸ and rFHR-5⁵⁹⁴ were mixed and the exchange reaction was monitored in time. Orange and green lines represent fits of a first-order exponential. Graphs are representative of n = 5. (D) Schematic overview of a monomer exchange experiment, to monitor reaction kinetics between previously equilibrated fluorescently labeled rFHRs and a >5 times molar excess of native rFHRs. Previously equilibrated FHR-2^488/594 (E) and FHR-5^488/594 (F) were mixed with an excess of native rFHR-1 (red), rFHR-2 (orange), rFHR-5 (green), or buffer control (blue). Graphs are representative of n ≥ 3.