Figure 2. Circular permutant GFP insertion topology reporters.

(A) the protein of interest X is inserted between the native N and C termini of GFP. Translation begins at an internal site in the GFP scaffolding (N′), so that the back of GFP is translated first. Correctly folded test inserts do not interfere with the folding and assembly of the GFP fragments. (B) Misfolded inserted target proteins prevent correct reconstitution of the two GFP fragments. (C) Truncations and new internal translation sites produce two independent fragments of GFP, reducing the amount of folded GFP. (D) Misfolded test protein fragments can sequester the fused GFP domain in aggregates preventing GFP assembly.