Fluorescence 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 dsDNA following its interaction with unlabelled proteins (BamHI,

A Quantitative Theoretical Framework For Protein-Induced Fluorescence Enhancement–Förster-Type Resonance Energy Transfer (PIFE-FRET)

“…Procedures and reagents are described in Ploetz and Lerner et al 11 The results analyzed here, were obtained from two different dsDNA systems as described before. In short, the distance dependence of PIFE in te absence of FRET was tested on a 40bp-long dsDNA 11 carrying Cy3(B) and ATTO 647N at opposite 5′-ends. All dsDNAs contained a palindromic sequence 5′-GGATCC-3′ for the restriction enzyme Bam HI placed at 1,2,3,5 or 7 bp away from the donor.…”

“…All dsDNAs contained a palindromic sequence 5′-GGATCC-3′ for the restriction enzyme Bam HI placed at 1,2,3,5 or 7 bp away from the donor. For experiments involving unspecific binding of T7 polymerase gp5/trx and controls for Bam HI, 45 bp-long dsDNA was used as described before 11 labeled with Cy3(B) at the 5′-end of the top strand, and ATTO 647N at 8,13,18,23,28 or 33bp of the bottom strand were employed.…”

Studying transcription initiation by RNA polymerase with diffusion‐based single‐molecule fluorescence

“…Moreover, Ploetz and Lerner et al . reached the same conclusion using sm experiments that simultaneously involved FRET and protein‐induced fluorescence enhancement (PIFE) measurements . These experiments utilized the Cy3 dye, which increases its fluorescence in response to a rise in local viscosity or steric restriction.…”

“…), DNA topology assays, magnetic tweezers, and sm fluorescence techniques (discussed in Ref. ). To sense conformational change in the transcription bubble during RP O formation, a modified diffusion‐based smFRET‐ALEX assay with a FRET dye pair positioned in the transcription bubble was used to conduct end‐point and real‐time monitoring experiments.…”

“…In particular, information about rare sub‐species, transient interactions between biomolecules, distinct subpopulations, or states for static or dynamic heterogeneous populations has been revealed through sm approaches . A variety of sm methods have been applied in the study of bacterial RNAP …”