Document Detail


Structural basis of fluorescence quenching in caspase activatable-GFP.
MedLine Citation:
PMID:  23139158     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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:
Samantha B Nicholls; Jeanne A Hardy
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2013-01-10
Journal Detail:
Title:  Protein science : a publication of the Protein Society     Volume:  22     ISSN:  1469-896X     ISO Abbreviation:  Protein Sci.     Publication Date:  2013 Mar 
Date Detail:
Created Date:  2013-02-25     Completed Date:  2013-08-29     Revised Date:  2014-03-07    
Medline Journal Info:
Nlm Unique ID:  9211750     Medline TA:  Protein Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  247-57     Citation Subset:  IM    
Copyright Information:
Copyright © 2013 The Protein Society.
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MeSH Terms
Descriptor/Qualifier:
Apoptosis
Blotting, Western
Caspases / genetics,  metabolism*
Chromatography, High Pressure Liquid
Circular Dichroism
Fluorescence*
Fluorescent Dyes / chemistry*,  metabolism
Genes, Reporter
Green Fluorescent Proteins / chemistry*,  genetics,  metabolism
Kinetics
Models, Molecular
Molecular Weight
Mutant Proteins / chemistry,  metabolism
Nuclear Magnetic Resonance, Biomolecular
Peptide Fragments / chemistry,  genetics,  metabolism
Protein Folding
Protein Structure, Secondary
Proteolysis
Recombinant Proteins
Spectrometry, Fluorescence
Grant Support
ID/Acronym/Agency:
GM080532/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Fluorescent Dyes; 0/Mutant Proteins; 0/Peptide Fragments; 0/Recombinant Proteins; 147336-22-9/Green Fluorescent Proteins; EC 3.4.22.-/Caspases
Comments/Corrections

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