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. 2013 Mar;22(3):247-57.
doi: 10.1002/pro.2188. Epub 2013 Jan 10.

Structural basis of fluorescence quenching in caspase activatable-GFP

Samantha B Nicholls  1 , Jeanne A Hardy
Affiliations

Structural basis of fluorescence quenching in caspase activatable-GFP

Samantha B Nicholls et al. Protein Sci. 2013 Mar.

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.

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Figures

Figure 1
Figure 1
A. CA-GFP is constructed from the Green Fluorescent Protein (S65T) GFP fused to a linker containing the caspase-3 and -7 recognition sequence DEVD to the 27 amino acid quenching peptide derived from the transmembrane domain of influenza M2. B. The structure of the GFP β-barrel (PDB ID:2YOG, green) with the chromophore residues shown as sticks. The 11th strand of the barrel, removed in the GFP1-10 construct is highlighted in purple. C. The CD spectra of GFP, CA-GFP and GFP1-10 are superimposed to allow assessment of the overall shape of the three spectra. The maximum intensity of the three spectra have been scaled for the relative concentration of each protein (Supporting Information Fig. S1). The non-scaled molar intensities of CD spectra for these proteins can be seen in Figure 2.
Figure 2
Figure 2
Circular dichroism spectra. A. The CD spectra of GFP from 200 to 250 nm collected at 5° increments from 20 to 90°C. The 20°C spectra is shown in the darkest shading; the 90°C spectra in the lightest shading and intermediate temperatures are shown in decreasing color intensity as a function of temperature. Spectra are plotted as the mean residual ellipticity (MRE × 103: degree cm2 dmol–1 number of residues–1) as a function of wavelength B. The CD spectra of CA-GFP collected and shown as in A. C. The CD spectra of GFP1-10 collected and shown as in A. D. Profile of thermal denaturation of GFP as monitored by CD signal at 214 nm plotted as a function of temperature. E. Profile of thermal denaturation of CA-GFP. F. Profile of thermal denaturation of GFP1-10.
Figure 3
Figure 3
The size exclusion chromatogram of GFP, CA-GFP, cleaved CA-GFP, and GFP1-10. GFP and cleaved CA-GFP elute at a retention volume consistent with a monomeric form of the protein. The monomer molecular weights are GFP: 27.7 kDa, CA-GFP: 32 kDa, cleaved CA-GFP: 28.2 kDa, and GFP1-10: 20.5 kDa. The dark CA-GFP elutes as two peaks with retention volumes consistent with a population that is 50% lower order oligomer (putatively trimer or tetramer) and 50% higher oligomer. GFP1-10 elutes in the void volume indicating a largely aggregated population.
Figure 4
Figure 4
Protease susceptibility of GFP, CA-GFP and GFP1-10. A. GFP, GFP1-10, and CA-GFP were subjected to digestion by proteinase K. Samples were collected and analyzed at 0, 1, 2, 5, and 10 min. GFP is highly resistant to digestion while GFP1-10 and CA-GFP are nearly completely degraded after 1 min. B. The GFP β-barrel (green) is drawn with the predicted caspase-6 cleavage sequence (VELD, blue) highlighted. The sequence falls in the center of the first β-strand, however, caspases are predicted to cleave in loop regions. C. GFP, GFP1-10, and CA-GFP were subjected to digestion by caspase-6. Caspase-6 is capable of cleaving GFP at a single site near the N-terminus. GFP is resistant to digestion while GFP1-10 and CA-GFP undergo partial cleavage after a two-hour incubation. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
Figure 5
Figure 5
TROSY NMR HSQC spectra. A. Spectra of GFP collected at 37°C. B. Spectra of CA-GFP also at 37°C. The spectra of GFP is indicative of a well-folded protein while the spectra of CA-GFP resembles that of an unfolded protein.
Figure 6
Figure 6
A. Constructs of CA-GFP and N-terminally tagged nCA-GFP with their relative fluorescence when co-expressed in E. coli with active caspase-7 or an inactive version caspase-7 C186A. CA-GFP shows a 40-fold increase in fluorescence in the presence of active caspase vs. nCA-GFP which shows less than 4-fold. B. Western blots of the CA-GFP and nCA-GFP co-transformations with active and inactive caspase. The top blot was probed with an anti-GFP primary antibody shows that CA-GFP is fully cleaved in the presence of active caspase while nCA-GFP is only partially cleaved. The bottom blot was probed with a primary antibody specific for the large sub-unit of caspase-7 shows that caspase-7 is cleaved and active in the case of the wild-type (WT) and in the full-length, unprocessed inactive form in the case of the C186A mutant. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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References

    1. Nicholls SB, Chu J, Abbruzzese G, Tremblay KD, Hardy JA. Mechanism of a genetically encoded dark-to-bright reporter of caspase activity. J Biol Chem. 2011;286:24977–24986. - PMC - PubMed
    1. I G, AD H, Regan L. Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J Am Chem Soc. 2000;122:5658–5659.
    1. Hu C, Chinenov Y, Kerppola T. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol Cell. 2002;9:789–798. - PubMed
    1. Cabantous S, Terwilliger TC, Waldo GS. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nature Biotech. 2004;23:102–107. - PubMed
    1. Wood TI, Barondeau Dp, Hitomi C, Kassmann CJ, Trainer JA, Getzoff ED. Defining the role of arginine 96 in green fluorescent protein fluorophore biosynthesis. Biochemistry. 2005;44:16211–16220. - PubMed

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