Enhanced Sensitivity of FRET‐Based Protease Sensors by Redesign of the GFP Dimerization Interface

Abstract

Real-time imaging of molecular events in living cells is important for understanding the basis of physiological processes and diseases. [1][2][3] Genetically encoded sensors that use fluorescence resonance energy transfer (FRET) [4] between two fluorescent proteins are attractive in this respect because they do not require cell-invasive procedures, can be targeted to different locations in the cell, and are easily adapted through mutagenesis and directed evolution approaches. [5][6][7] Following the pioneering work of Roger Tsien and others, on genetically encoded protease and calcium sensors, FRET-based imaging probes have been developed for many other small molecules and cell signaling events. [8][9][10][11][12][13] In these probes, conformational changes in a sensor domain are translated into a change in energy-transfer efficiency between donor and acceptor fluorescent domains, which is detected by a change in the ratio of donor and acceptor emission. This ratiometric response is independent of the sensor concentration, which is an important advantage of FRET-based sensors. In practice, however, most FRET-based sensors display only a relatively small difference in emission ratio upon activation. Improvement of these ratiometric changes has been recognized as an important prerequisite for use of these sensor systems in high-throughput applications based on fluorescence plate readers and fluorescence assisted cell sorting (FACS). [14,15] Recently a pair of CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein) variants, CyPet and YPet, respectively, have been reported that were optimized for FRET through a process of directed evolution.[16] When incorporated in a protease sensor, a 20-fold change in emission ratio was observed upon cleavage of a flexible peptide that linked CyPet and YPet, compared to only a fourfold change for the same construct with enhanced CFP (ECFP) and enhanced YFP (EYFP) domains. However, the mechanism behind their remarkable FRET properties has remained unclear. A total of eighteen mutations were introduced in the course of their development, many of which were at the exterior of the protein, at a large distance from the fluorophore. Moreover, no large differences in quantum yield or extinction coefficient were reported; this suggests that the photophysical properties of the fluorescent proteins were not significantly altered. We therefore hypothesized that the increase in FRET observed for CyPet and YPet could be due to an enhanced tendency to interact when connected by a peptide linker. The parent green fluorescent protein (GFP) has a known tendency to dimerize, [17] and analysis of the mutations in YPet have identified two residues, S208F and V224L, that are present at the dimer interface, as shown by the X-ray structure of the GFP dimer. Here, we show that A C H T U N G T R E N N U N G introduction of just these two mutations in both fluorescent domains of ECFP-linker-EYFP constructs results in a fourfold increase in the EYFP-to-ECFP emission ratio, which yields a 16-fol...

Advances in fluorescent protein technology

“…The resistance to acidic environments afforded by YPet is superior to Venus and other YFP derivatives, which will enhance the utility of this probe in biosensor combinations targeted at acidic organelles. Although optimized for FRET, there remains a serious doubt as to the origin of YPet's increased performance, which is likely to be due simply to enhanced dimerization with its co-evolved partner CyPet (Ohashi et al, 2007;Vinkenborg et al, 2007).…”

“…The CyPet-YPet FRET caspase-3 biosensor exhibits a 20-fold improvement in dynamic range over a similar mCeruleanmVenus pair. This impressive increase in efficiency has recently been challenged (Ohashi et al, 2007;Vinkenborg et al, 2007) by demonstrations that CyPet-YPet biosensors are prone to enhanced dimerization, which is largely responsible for their superior performance. Thus, careful consideration to this artifact should be given in the design of experiments using the CyPet-YPet combination.…”

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

“…This effect may be due to reporter dimerization at high nanomolar to micromolar concentrations where intermolecular FRET, in addition to concentrationindependent intramolecular FRET, contributes to the overall FRET fluorescence emission. Indeed, some green fluorescent protein (GFP) derivatives exhibit concentration-dependent dimerization [35,36]. These nonlinear effects have consequences for assay precision because errors in reporter dilution can lead to changes in the ratiometric value of the assay, thereby reducing accuracy.…”

Rational Design of FRET-Based Sensor Proteins

“…Although wild-type ECFP and EYFP do not interact when connected by flexible SGGSGG linkers, we showed that subtle redesign of the dimerization interface by substitution of the hydrophilic Ser208 by a hydrophobic phenylalanine promotes intramolecular complex formation between the two fluorescent domains, resulting in a large increase in energy transfer (Fig. 12) [21]. The introduction of a second mutation at the dimerization interface, V224L, did not by itself enhance intramolecular interactions between ECFP and EYFP, but it did induce a further increase in energy transfer efficiency in the presence of the S208F mutation.…”

“…Variants of ECFP and EYFP, CyPet and YPet, were identified that, when linked via a flexible caspase recognition sequence, yielded a FRET sensor with a 20-fold ratiometric change upon cleavage by caspase-3. Although YPet and CyPet have been advertised as the donor and fluorescent domains of choice for FRET sensors [18,19], we and others have recently shown that their improved FRET properties are caused by an increased tendency to form an intramolecular complex when linked via a flexible peptide linker, limiting their potential as general applicable fluorescent domains for FRET sensors [20,21].…”