Protein-Induced Long Lifetime Luminescence of Nonmetal Probes

Abstract

Time-resolved luminometry-based assays have great potential for measurements in complicated biological solutions and living cells as the measured signal can be easily distinguished from nanosecond lifetime background fluorescence of organic compounds and autofluorescence of cells. In the present study we discovered that binding of a thiophene- or a selenophene-containing heteroaromatic moiety (luminescence donor) to the purine-binding pocket of a protein kinase (PK) induces long lifetime photoluminescence signal that is largely intensified through efficient energy transfer to a fluorescent dye present in close proximity to the luminescence donor. The developed ARC-Lum probes possessing 19-266 μs luminescence lifetime when associated with the target kinase can be used for determination of activity of basophilic PKs, characterization of inhibitors of PKs, and as cAMP sensors. An ARC-Lum probe was also used for the determination of kinetic parameters of inhibitor binding to the catalytic subunit of protein kinase A (PKAc). Effective real-time monitoring of the activation of PKA by Forskolin and the displacement of an ARC-Lum probe from its complex with PKA by inhibitor H89 was performed in live cells. The discovered phenomenon, protein-induced long lifetime luminescence of aromatic probes is very likely to occur with all PKs and many other proteins.

Deoxygenation Increases Photoluminescence Lifetime of Protein-Responsive Organic Probes with Triplet–Singlet Resonant Energy Transfer

“…9,11 The signal intensity of the microsecond-lifetime luminescence of ARC-Lum(Fluo) probes in complex with a PK is 20-to 1000-fold higher than that of the phosphorescence signal of their ARC-Lum(−) counterparts. 9 This enhancement is bigger for thiophenecontaining compounds than for selenophene-containing compounds whereas the selenophene-containing ARC-Lum probes are brighter in complex with PKs than their thiophenecontaining counterparts. 9 A cascade mechanism of energy transfer ( Figure 3) that was proposed for the discovered phenomenon included efficient Forster-type resonant energy transfer (FRET) from the excited triplet state of the low-QY donor phosphor ( 3 D*) to the singlet state of the acceptor fluorophore ( 1 A), possessing high QY.…”

“…9 This enhancement is bigger for thiophenecontaining compounds than for selenophene-containing compounds whereas the selenophene-containing ARC-Lum probes are brighter in complex with PKs than their thiophenecontaining counterparts. 9 A cascade mechanism of energy transfer ( Figure 3) that was proposed for the discovered phenomenon included efficient Forster-type resonant energy transfer (FRET) from the excited triplet state of the low-QY donor phosphor ( 3 D*) to the singlet state of the acceptor fluorophore ( 1 A), possessing high QY. 9 This energy transfer leads to the excited singlet state of the acceptor ( 1 A*), which thereafter emits light, resulting in powerful sensitization of the phosphorescence emission.…”

“…9 A cascade mechanism of energy transfer ( Figure 3) that was proposed for the discovered phenomenon included efficient Forster-type resonant energy transfer (FRET) from the excited triplet state of the low-QY donor phosphor ( 3 D*) to the singlet state of the acceptor fluorophore ( 1 A), possessing high QY. 9 This energy transfer leads to the excited singlet state of the acceptor ( 1 A*), which thereafter emits light, resulting in powerful sensitization of the phosphorescence emission. 9 Long lifetime of the emission mediated by the short-lifetime fluorescent dye is due to slow resonant energy transfer from 3 D* to 1 A leading to excitation of the dye to 1 A* and relaxation of 3 D* to the ground singlet state ( 3 D* + 1 A → 1 D + 1 A*).…”

Slowly on, Slowly off: Bisubstrate‐Analogue Conjugates of 5‐Iodotubercidin and Histone H3 Peptide Targeting Protein Kinase Haspin

“…Next, we established an assay format to assess the on‐ and off‐rates of the compounds that were not fluorescently labelled, including 1 and 5‐ITu. We took advantage of our PK binding‐responsive photoluminescent bisubstrate probe ARC‐1063, which gives a very low background signal as a free probe, but features strong, long‐lifetime (microsecond scale) photoluminescence at 647 nm (80–480 μs after pulse‐excitation at 337 nm) when complexed with a target PK . Importantly, because of the long photoluminescence lifetime of the ARC‐1063/PK complex, the probe could also be used for measurements in assay mixtures containing fluorescent compounds that do not exhibit such long‐lifetime photoluminescence (hence do not affect read‐out).…”

Cover Picture: Modulable and Highly Diastereoselective Carbometalations of Cyclopropenes (Chem. Eur. J. 4/2014)

“…The displacement experiments (Figure 2 a and figures S2 and S3 in the Supporting Information) were performed at relatively high concentration of the ARC–Lum probe ARC‐1139 (total concentration 100 n M ) compared with that of Pim‐1 (2 n M ). This was needed for shifting the displacement IC 50 curves to higher concentration values for characterization of tight‐binding inhibitors 16. As ARC(PIM) inhibitors have relatively low affinity toward PKAc, ARC‐1188 at a concentration of 5 n M was used for displacement experiments with PKAc (2 n M ).…”