PIFE meets FRET-a molecular ruler goes 2D

E. Ploetz, E. Lerner, F. Husada, M. Roelfes, S. Y. Chung, J. Hohlbein, S. Weiss, .

Research output: Working paper / PreprintPreprint


Advanced microscopy methods allow to obtain information on (dynamic) conformational changes in biomolecules via the measure of a single molecular distance in the structure. It is, however, extremely difficult to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using a one-dimensional spatial measure. In this paper we address this fundamental problem by extending the standard ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can report on e.g., the state of dsDNA and its interaction with unlabelled proteins (BamHI, EcoRV, T7 DNA polymerase p5/trx). The PIFE-FRET assay uses established labelling protocols and fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules.
Original languageEnglish
Number of pages31
StatePublished - 2016


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