Fluorescence‐Lifetime‐Sensitive Probes for Monitoring ATP Cleavage

Abstract

Adenosine triphosphate (ATP) probes modified with fluorescence dyes that change their fluorescence properties upon cleavage are an interesting tool for monitoring enzymatic ATP turnover. As a readout parameter, fluorescence lifetime is attractive because it is nearly independent of concentration. In our study, we synthesised and investigated fifteen different ATP analogues, in which the fluorophores were attached to the γ-phosphate of ATP. All analogues showed distinctly different fluorescence lifetimes compared to the corresponding values of the free fluorophores. Both increases and decreases in fluorescence lifetime were observed upon attachment to ATP. To shed light on the photophysical processes governing the lifetime changes, we performed photoelectron spectroscopy in air (PESA) to determine HOMO energy levels and time-resolved fluorescence spectroscopy to obtain rate constants. We present evidence that fluorescence quenching in the compounds tested is dynamic and attributed to photoinduced electron transfer (PET), whereas fluorescence lifetime increases are caused by stacking interactions between chromophore and the nucleobase reducing non-radiative relaxation. Finally, we demonstrate that enzymatic cleavage of the ATP analogues presented can be followed by continuous monitoring of fluorescence lifetime changes.

Fluorescently Labelled ATP Analogues for Direct Monitoring of Ubiquitin Activation

“…To develop new suitable dye‐adenine pairs, we used photoelectron spectroscopy in air (PESA) to determine HOMO energy levels for hydrophilic dyes. With this approach, we recently were able to investigate different fluorophores like rhodamines and BODIPYs from dry thin films that suited for fluorescence lifetime readout when attached to ATP . While we could demonstrate enzymatic processing and real‐time detection of these compounds, their main drawback was the relatively short lifetime of these dyes.…”

“…Similar to UBA1, UBA6 is known to support E6AP autoubiquitination . Hence, to determine the ability of UBA6 to employ our ATP analogues, we also followed the formation of polyubiquitinated E6AP.…”

“…Compared to other probes, these compounds contain only one instead of two fluorophores, which promises better enzymatic acceptance. Fluorescence lifetime changes between the intact and the cleaved ATP analogues are caused by the quenching of fluorescence by the nucleobase adenine [16] (Figure 1 B). The quenching is caused by photoinduced electron transfer (PET).…”

“…PET-based quenchers that have previously been employed in biological assays are guanine (À5.33 eV; calculated from its redox potential) [20] and tryptophan (À4.90 eV; calculated from its redox potential), [21] both of which possess high HOMO energies. [22][23][24][25][26] By contrast, due to its lower HOMO energy (À5.78 AE 0.01 eV), [16] adenine has only rarely been used as quencher. To develop new suitable dye-adenine pairs, we used photoelectron spectroscopy in air (PESA) to determine HOMO energy levels for hydrophilic dyes.…”

“…With this approach, we recently were able to investigate different fluorophores like rhodamines and BODIPYs from dry thin films that suited for fluorescence lifetime readout when attached to ATP. [16] While we could demonstrate enzymatic processing and real-time detection of these compounds, their main drawback was the relatively short lifetime of these dyes. This made the detection of lifetime changes especially under biologically relevant conditions difficult.…”