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Homogeneous TR-FRET High-Throughput Screening Assay for Calcium-Dependent Multimerization of Sorcin
Heidi Appelblom, MSc
Tykistokatu 6 A BioCity 6th Floor 20520 Turku, Finland, heidi.appelblom{at}utu.fi
Jussi Nurmi
University of Turku, Department of Biotechnology, Finland
Tero Soukka
University of Turku, Department of Biotechnology, Finland
Michael Pasternack
Orion Pharma, Espoo, Finland
Kai E. Penttilä
Orion Pharma, Espoo, Finland
Timo Lövgren
University of Turku, Department of Biotechnology, Finland
Pauliina Niemelä
University of Turku, Department of Biotechnology, Finland
A homogeneous high-throughput screening method based on time-resolved fluorescence resonance energy transfer (TR-FRET) for the measurement of calcium-dependent multimerization of an EF-hand protein, sorcin, is described. The assay is based on a specific sorcin binding peptide conjugated either with an intrinsically highly fluorescent europium chelate (donor) or an Alexa Fluor 700 fluorophore (acceptor). Addition of calcium results in multimerization of sorcin, allowing several peptides to bind simultaneously to the epitopes of the multimeric protein complex, and the proximity of peptides labeled either with donor or acceptor label results in fluorescence resonance energy transfer between the 2 labels. When no calcium is present, the protein remains in a monomer form, and thus no FRET can take place. In the optimized assay construct, the assay was performed in 45 min, and a more than 20-fold signal-to-background ratio was achieved. The reversibility of sorcin multimerization was shown by chelating free calcium with ethylenediamine tetraacetic acid (EDTA). The developed homogeneous assay can be used in screening molecules that either inhibit or enhance multimerization of sorcin, and the assay format is applicable to various noncompetitive high-throughput screening assays detecting protein multimerization reactions. (Journal of Biomolecular Screening 2007:842-848)
Key Words: time-resolved fluorescence resonance energy transfer • homogeneous assay • sorcin • multimerization
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This version was published on September
1, 2007
Journal of Biomolecular Screening, Vol. 12, No. 6,
842-848 (2007)
DOI: 10.1177/1087057107303973
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