Mechanism of a Genetically Encoded Dark-to-Bright Reporter for Caspase Activity

Abstract

Fluorescent proteins have revolutionized modern biology with their ability to report the presence of tagged proteins in living systems. Although several fluorescent proteins have been described in which the excitation and emission properties can be modulated by external triggers, no fluorescent proteins have been described that can be activated from a silent dark state to a bright fluorescent state directly by the activity of an enzyme. We have developed a version of GFP in which fluorescence is completely quenched by appendage of a hydrophobic quenching peptide that tetramerizes GFP and prevents maturation of the chromophore. The fluorescence can be fully restored by catalytic removal of the quenching peptide, making it a robust reporter of proteolysis. We have demonstrated the utility of this uniquely dark state of GFP as a genetically encoded apoptosis reporter that monitors the function of caspases, which catalyze the fate-determining step in programmed cell death. Caspase Activatable-GFP (CA-GFP) can be activated both in vitro and in vivo, resulting in up to a 45-fold increase in fluorescent signal in bacteria and a 3-fold increase in mammalian cells. We used CA-GFP successfully to monitor real-time apoptosis in mammalian cells. This dark state of GFP may ultimately serve as a useful platform for probes of other enzymatic processes.

Structural basis of fluorescence quenching in caspase activatable‐GFP

“…We have previously shown that the appendage of the 27-amino acid quenching peptide causes CA-GFP to oligomerize into a mixed population that is predominantly tetramer and higher order oligomers. 1 We hypothesize that this oligomerization is at the root of the CA-GFP quenching mechanism. To fully harness the potential of CA-GFP for other uses, it is essential to understand the structural basis of the immature chromophore at a mechanistic level.…”

“…13,14 Despite the fact that all 11 strands must be available for proper folding of GFP, it is possible to cut GFP following the 10th strand to make split GFP. 4 In the split-GFP system, co-expression of GFP strands 1-10 (GFP [1][2][3][4][5][6][7][8][9][10] ) with the 11th strand allows reassociation of the 11th strand with GFP [1][2][3][4][5][6][7][8][9][10] and folding of the GFP b-barrel. Once the full GFP b-barrel is formed the chromophore is competent to mature, and split GFP becomes fluorescent.…”

“…Chromophore maturation in split GFP occurs with similar kinetics to CA-GFP. 4,15 We hypothesized that CA-GFP might adopt a stable, partially folded structure similar to that of GFP [1][2][3][4][5][6][7][8][9][10] [ Fig. 1(C)].…”

“…Indeed, the overall shape of the GFP 1-10 spectrum was also indicative of a predominantly b-strand structure. More importantly, the CD spectra for GFP [1][2][3][4][5][6][7][8][9][10] and CA-GFP are more similar to each other than to mature GFP. This suggests a higher degree of structural similarity between GFP 1-10 and CA-GFP than mature GFP.…”

“…We recently reported the development of a genetically-encoded reporter of caspase activity called caspase activatable-GFP (CA-GFP) 1 that has many advantageous properties. The genetically-encoded characteristic of CA-GFP makes it amenable to a number of applications that cannot be fulfilled by existing reporters.…”