Apoptosis is critical for organismal homeostasis and a wide variety of diseases. 
Caspases are the ultimate executors of the apoptotic programmed cell death 
pathway. As caspases play such a central role in apoptosis, there is significant 
demand for technologies to monitor caspase function. We recently developed a 
caspase activatable-GFP (CA-GFP) reporter. CA-GFP is unique due to its "dark" 
state, where chromophore maturation of the GFP is inhibited by the presence of a 
C-terminal peptide. Here we show that chromophore maturation is prevented 
because CA-GFP does not fold into the robust β-barrel of GFP until the peptide 
has been cleaved by active caspase. Both CA-GFP and GFP₁₋₁₀ , a split form of 
GFP lacking the 11th strand, have similar secondary structure, different from 
mature GFP. A similar susceptibility to proteolytic digestion indicates that 
this shared structure is not the robust, fully formed GFP β-barrel. We have 
developed a model that suggests that as CA-GFP is translated in vivo it follows 
the same folding path as wild-type GFP; however, the presence of the appended 
peptide does not allow CA-GFP to form the barrel of the fully matured GFP. 
CA-GFP is therefore held in a "pro-folding" intermediate state until the peptide 
is released, allowing it to continue folding into the mature barrel geometry. 
This new understanding of the structural basis of the dark state of the CA-GFP 
reporter will enable manipulation of this mechanism in the development of 
reporter systems for any number of cellular processes involving proteases and 
potentially other enzymes.