Deep Quench:  An Expanded Dynamic Range for Protein Kinase Sensors

Abstract

Assays that furnish a fluorescent readout of protein kinase activity provide a means to identify and characterize inhibitory agents, assess structure-function relationships, and correlate enzyme activity with cellular behavior. Although several protein kinase sensors have been described in the literature, their fluorescent response to phosphorylation are generally modest to moderate (1.1 -8-fold). We have developed a "deep quench" strategy that elicits a dramatic amplification of fluorescence (>60-fold) in cAMP-dependent protein kinase substrates. We describe sensor design, assay development via a combination of library synthesis and screening, characterization of the assay components, and an assessment of two inhibitory species.Protein kinases catalyze the phosphorylation of serine, threonine, and tyrosine residues in protein and peptide substrates. These enzymes have received considerable attention due to the relationship between aberrant kinase activity and an assortment of human afflictions. Specific and highly sensitive protein kinase sensors furnish a means to rapidly identify inhibitors, assess protein structure/function relationships, and correlate kinase activity with cellular behavior. A large number of kinase assays have been described, however, assays with fluorescent readouts are most easily applied to both in vitro and intracellular settings. GFP-labeled protein and fluorophore-labeled peptide substrates generally deliver, upon phosphorylation, a fluorescent response that varies from 10-60% to 2-9-fold, respectively. 1 By comparison, many fluorescent sensors developed for a variety of biomolecules (e.g. proteinases 2 and the detection of specific nucleotide sequences 3 ) display enhancements of 25-fold and greater. A large dynamic range offers enhanced sensitivity, thereby furnishing a means to assess target biomolecule behavior under a variety of conditions. Unlike nearly all of the protein kinase assays reported to date, 4 the readout described in studies with proteinases 2 and molecular beacons 3 arise via relief of fluorescent quenching. We report herein an approach, devised around the latter conceptual framework ("Deep Quench"), which delivers a robust protein kinase-elicited fluorescent response.Our initial studies have focused on the strategy outlined in Scheme 1. A fluorophore-labeled kinase substrate (A) exhibits little or no fluorescence (B) in the presence of a quencher molecule. Upon phosphorylation, the peptide product (C) is sequestered by a phospho-Ser binding domain to form the complex D, which disrupts the interaction between peptidefluorophore and quencher. The latter should partially or completely restore the fluorescence of the starting peptide.Pyrene was chosen to serve as the fluorophore on an amino acid sequence (AcGRTGRRFSYPamide) recognized by the cAMP-dependent protein kinase ("PKA"). 5 We employed the phospho-Ser binding domain, 14-3-3τ, to serve as the sequestering agent since 14-3-3 domains display a high affinity for phosphoSer-containing peptides (K D < 100 nM...

Fluorescent Biosensors of Intracellular Targets from Genetically Encoded Reporters to Modular Polypeptide Probes

“…Therefore in an attempt to control and optimize the sensitivity and selectivity of the response so as to obtain a robust fluorescent signal upon target recognition, strategies have been devised to quench and/or cage the fluorophore. Strategies of intramolecular fluorophore quenching have proven to be effective in reducing background fluorescence prior to target binding to fluorescent peptide biosensors both in vitro and in living cells [73][74][75][76]. Molecular caging allows the conversion of a biomolecule into an inert form through chemical modification with a photolabile protective group.…”

Peptide-based fluorescent sensors of protein kinase activity: Design and applications

“…In this particular case, the PKA substrate library was constructed using amine-handles at seven different positions along the consensus sequence, to which various fluorophores were coupled (see Figure 2B). The lead pyrene-based species displays a 1.6-fold phosphorylation-induced enhancement in fluorescence [6]. The modest fluorescent response in the PKA/14-3-3 system may be a consequence of the fluorophore's inability to access a hydrophobic subsite.…”

“…A screening-based approach was used with 47 potential quenchers in conjunction with the pyrene flurophore, that was covalently appended to ten different sites on PKA consensus peptides [6]. We employed the 14-3-3 domain as a phosphopeptide-capture motif whose presence is designed to relieve pyrene fluorescence quenching by sequestering the phosphorylated peptide (see Figure 3).…”

“…For example, in a single reaction cuvette, a continuous "Deep-Quench" sensor was employed to report the cGMP-dependent activation of the cGMP dependent protein kinase (see Figure 5B and Ref. [6]). In addition, these fluorescent sensing systems can be used to examine both peptide-based and ATP-site-directed inhibitors ( Figure 5C) under conditions that mimic the intracellular milieu (i.e.…”

“…In addition, these fluorescent sensing systems can be used to examine both peptide-based and ATP-site-directed inhibitors ( Figure 5C) under conditions that mimic the intracellular milieu (i.e. physiological concentrations of ATP) [6]. For example, the "Self Reporting" sensor proved to be exceptionally useful in demonstrating the striking selectivity displayed by an active-site directed bivalent inhibitor between a subset of kinases [9].…”