Abstract
Simple and robust assays to monitor enzymatic ATP cleavage with high efficiency in real‐time are scarce. To address this shortcoming, we developed fluorescently labelled adenosine tri‐, tetra‐ and pentaphosphate analogues of ATP. The novel ATP analogues bear — in contrast to earlier reports — only a single acridone‐based dye at the terminal phosphate group. The dye's fluorescence is quenched by the adenine component of the ATP analogue and is restored upon cleavage of the phosphate chain and dissociation of the dye from the adenosine moiety. Thereby the activity of ATP‐cleaving enzymes can be followed in real‐time. We demonstrate this proficiency for ubiquitin activation by the ubiquitin‐activating enzymes UBA1 and UBA6 which represents the first step in an enzymatic cascade leading to the covalent attachment of ubiquitin to substrate proteins, a process that is highly conserved from yeast to humans. We found that the efficiency to serve as cofactor for UBA1/UBA6 very much depends on the length of the phosphate chain of the ATP analogue: triphosphates are used poorly while pentaphosphates are most efficiently processed. Notably, the novel pentaphosphate‐harbouring ATP analogue supersedes the efficiency of recently reported dual‐dye labelled analogues and thus, is a promising candidate for broad applications.
Highlights
Ubiquitination — the covalent modification of proteins by the 76 amino acid protein ubiquitin (Ub) — is a ubiquitous protein modification with fundamental roles in numerous cellular processes including protein degradation, DNA damage repair, cell cycle regulation and gene expression.[1,2] Malfunction of the ubiquitination system contributes to a broad variety of human diseases like cancer, diabetes or neurodegenerative disorders.[3,4] For the attachment of Ub to substrate proteins, the consecutive action of at least three classes of enzymes is needed
We have recently developed a time-resolved ATPase sensor (TRASE) based on a Fçrster resonance energy transfer (FRET) pair embedded within the ATP scaffold to continuously monitor ATP-dependent enzymes.[13,14]
The quenching is caused by photoinduced electron transfer (PET)
Summary
Ubiquitination — the covalent modification of proteins by the 76 amino acid protein ubiquitin (Ub) — is a ubiquitous protein modification with fundamental roles in numerous cellular processes including protein degradation, DNA damage repair, cell cycle regulation and gene expression.[1,2] Malfunction of the ubiquitination system contributes to a broad variety of human diseases like cancer, diabetes or neurodegenerative disorders.[3,4] For the attachment of Ub to substrate proteins, the consecutive action of at least three classes of enzymes is needed. Available E1 activity assays include SDS-PAGE analysis of E1/E2-Ub thioester conjugates by Western Blot,[8] radio-labelling of the involved proteins with 125[I][9] or 32[P],[10] FRET between Ub and E1[11] or enzyme-coupled spectrophotometric assays for phosphate determination.[12] Drawbacks of these assays are that they are either laborious, do not allow continuous read-out or are dependent on additional enzymes of the downstream cascade To fill this gap, we have recently developed a time-resolved ATPase sensor (TRASE) based on a Fçrster resonance energy transfer (FRET) pair embedded within the ATP scaffold to continuously monitor ATP-dependent enzymes.[13,14] This ATP FRET probe sensor uses two fluorescent dyes, a donor and an acceptor dye, that are attached to the terminal phosphate group and to adenine, respectively. Elongation of the phosphate chain to pentaphosphate resulted in even better analogues enabling us to follow the activation of Ub by both enzymes, UBA1 and UBA6, in real-time
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