New Thiol-Reactive Eu-Complex for Distance Measurements by LRET

Crystallography and NMR spectroscopy are ideally suited to resolve the 3D structures of biomolecules but the material and time demand for each structure is high. Fluorescence resonance energy transfer (FRET) provides less structural information but is better suited to study conformational changes and structure-function relationships by screening a large number of mutants or experimental conditions. Moreover, FRET allows for real time monitoring of conformational changes induced by specific ligands. Usually, FRET yields only a crude estimate of the donor-acceptor distance, due to the fact that the relative orientation of donor and acceptor are rarely known. Luminescence resonance energy transfer (LRET) is much better suited for distance measurements because the orientation factor (and thus the Forster distance) are known, and because energy transfer is measured by a change of lifetime, rather than of signal intensity. In LRET-experiments the ideal donors are highly stable Eu/Tb‑complexes, with a single lifetime that is not influenced by attaching the complex to a biomolecule. Several terpyridine-based Eu-complexes described in literature have promising properties concerning uniform lifetimes after protein labeling but all described complexes have rather long linkers which prevent accurate distance measurements. In this study, a new terpyridine-based Eu-complex with maleimide very close to the metal ion center was synthesized and found to have ideal properties for distance measurement by LRET: After linking to the single cysteine of BSA, the complex showed a quantum yield of 30%, a single lifetime of 1.2 ms, comparable to the best known Eu-complexes, and the lifetime was unaffected by phosphate or EDTA. In conclusion, this new Eu complex appears ideally suited for reliable measurement of intra- or intermolecular distances.The work was supported by grant W0125 of the Austrian science fund (FWF)