title: Structural basis of fluorescence quenching in caspase activatable-GFP.
abstract: 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.
authors.affiliation: Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts 01003, USA.
authors.forename: Samantha B
authors.initials: SB
authors.lastname: Nicholls
authors.forename: Jeanne A
authors.initials: JA
authors.lastname: Hardy
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D059748:Proteolysis; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence
chemical_list: D005456:Fluorescent Dyes; D050505:Mutant Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D020169:Caspases