Crystallography and NMR spectroscopy are ideally suited to resolve the 3D structures of biomolecules, but the material and time demand for each molecular structure is high. Fluorescence resonance energy transfer (FRET) provides less structural information but it is better suited for studying 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 Förster distance) is known, and because energy transfer is measured by a change of the donor's lifetime, rather than of its signal intensity. ^In LRET-experiments the ideal donors are highly stable Eu- or Tbcomplexes, 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 thesis, new terpyridine-based Eucomplexes with very short linkers were synthesized and characterized. Complexes with two kinds of linkers designed for site-specific labeling were synthesized: (I) A maleimide-linker provided for coupling to thiols or cysteine mutants and (II) hydrazide-linker for coupling to aldehyde- or ketogroups or N-terminal serines/threonines after oxidation with periodate. ^The complexes showed a high quantum yield (32%), a single long lifetime (1.25 ms) which was not influenced by coupling to protein, very high stability in the presence of chelators like EDTA or EGTA, and no interaction with cofactors like ATP, ADP, AMP or GTP, as required for application to practical problems. LRET was measured in different model systems like DNA, coiled coil-forming peptides, and proteins. A variety of problems was encountered in these model studies: The synthetic DNA strands were only available with amino- or thiohexyl-linkers which obviated accurate distance measurement. Oligomerization or higher forms of aggregation led to quenching of one donor by more than one acceptor, and in some cases a direct interaction of donor and acceptor dye was observed. These findings revealed the difficulties in finding a well-defined calibration system for LRET. Fortunately the distance measurement by LRET was seen to properly work in SecA dimers. ^As described in the literature, the experiments with SecA showed that dimerization was dependent on the salt concentration in the buffer. Furthermore, LRET proved to be ideally suited to find out which surface areas in SecA-dimers are interacting with each other. This result is an example of scientific problems where LRET can give a correct answer, whereas crystallography cannot.