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 b-barrel of GFP until the peptide has been cleaved by active caspase. Both CA-GFP and GFP 1-10 , 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 b-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.

A dark-to-bright reporter cell for classical swine fever virus infection

“…Therefore, compared to ELISA, RT-PCR or immunocytochemistry techniques using fixed cells, the likelihood of artifacts is reduced and its activity is easily detectable using a fluorescence microscope. GFP has a typical beta barrel structure, consisting of eleven b-sheets with six alpha helix(s) and the beta barrel structure is a nearly perfect cylinder (Ormö et al, 1996;Nicholls and Hardy, 2013). GFP is nonfluorescent until the protein is folded properly to form a chromophore by post-translational modification (Pouwels et al, 2008).…”

“…GFP is nonfluorescent until the protein is folded properly to form a chromophore by post-translational modification (Pouwels et al, 2008). Fusing the quenching peptide to the GFP C-terminus may prevent proper folding of GFP, resulted a dark state with an immature chromophore until the quenching peptide is cleaved and released (Nicholls and Hardy, 2013).…”

“…It was reported that fluorescent proteins can be activated from a silent dark state to a bright fluorescent state directly by the activity of an enzyme (Mahajan et al, 1999;Nicholls et al, 2011;Nicholls and Hardy, 2013). Chromophore maturation of GFP is prevented because the GFP protein does not fold into the correct pattern until the quenching peptide has been specifically cleaved by certain enzyme (Nicholls and Hardy, 2013).…”

“…Chromophore maturation of GFP is prevented because the GFP protein does not fold into the correct pattern until the quenching peptide has been specifically cleaved by certain enzyme (Nicholls and Hardy, 2013). The aim of this study was to develop a cell-based EGFP reporter system, in which fluorescence is fully quenched by a short peptide.…”

Live Cell Reporter Systems for Positive-Sense Single Strand RNA Viruses

“…This assay was based on a novel reporter cell stably expressing EGFP fused in-frame to a quenching peptide via a special recognition sequence of the CSFV NS3 protease [11]. Without the CSFV infection, Although EGFP can be expressed in the reporter cell, chromophore maturation of EGFP in the reporter cells was inhibited by the C-terminal quenching peptide of EGFP [56]. However, when the cells were infected with CSFV, the recognition sequence of the CSFV NS3 protease was specifically cleaved by the protease, and the quenching peptide was released from the protein.…”