Split PAmCherry1 for BiFC.
(A) Crystal structure of PAmCherry1 with the 159/160 split site for BiFC indicated. The site is located between beta sheets 7 and 8; (B) Artificial dimerization system for testing PAmCherry1 BiFC. Non-fluorescent PAmCherry1 fragments are fused to dimerization domains, DmrA and DmrC. The N-terminus of DmrA has a myristoylation signal that localizes it to the plasma membrane. DmrA/DmrC heterodimerization is induced with the addition of a small molecule heterodimerizer, bringing the PAmCherry1 fragments together to reconstitute an intact PAmCherry1; (C) Four configurations were tested for BiFC with the inducible heterodimerization system. A flexible (GGGGS)2 linker was used for all constructs. RN = PAmCherry1 N-terminal fragment (residues 1–159), RC = PAmCherry1 C-terminal fragment (residues 160–236); (D) Total internal reflection fluorescence (TIRF) images of heterodimerizer induced BiFC signals in DmrA-RC/DmrC-RN (top right panel) and DmrA-RC/RN-DmrC (bottom right panel). Constructs were transiently transfected into U2OS cells and cells on the right were exposed to 500 nM heterodimerizer overnight prior to imaging; (E) Average BiFC signal intensities at four different split sites: 158, 159, 160 and 174 (n = 5–10). Intensities are normalized to split site 159. Error bars are SEM. Scale bars, (D) 10 µm.