title: Förster resonance energy transfer and protein-induced fluorescence enhancement as synergetic multi-scale molecular rulers.
abstract: Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular 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 simultaneously report on e.g., the conformational state of double stranded DNA (dsDNA) following its interaction with unlabelled proteins (BamHI, EcoRV, and T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule 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. Finally, we study transcription initiation and scrunching of E. coli RNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble.
authors.affiliation: Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Evelyn
authors.initials: E
authors.lastname: Ploetz
authors.affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Eitan
authors.initials: E
authors.lastname: Lerner
authors.affiliation: Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Florence
authors.initials: F
authors.lastname: Husada
authors.affiliation: Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Martin
authors.initials: M
authors.lastname: Roelfs
authors.affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: SangYoon
authors.initials: S
authors.lastname: Chung
authors.affiliation: Laboratory of Biophysics, Wageningen University and Research, 6708 WE, Wageningen, The Netherlands.|Microspectroscopy Centre, Wageningen University and Research, 6708 WE, Wageningen, The Netherlands.
authors.forename: Johannes
authors.initials: J
authors.lastname: Hohlbein
authors.affiliation: Department of Chemistry and Biochemistry, University of California Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Shimon
authors.initials: S
authors.lastname: Weiss
authors.affiliation: Molecular Microscopy Research Group, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
authors.forename: Thorben
authors.initials: T
authors.lastname: Cordes