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.
Highlights
This restriction prohibits monitoring an essential feature of biological processes
We utilize the stoichiometry parameter, S, as a measure for protein-induced fluorescence enhancement (PIFE)-effects, while Förster resonance energy transfer (FRET) reports on the distance between fluorophores
For this we suggest to use alternating laser excitation (ALEX), which reports on PIFE effects via the ratiometric parameters[51], stoichiometry, S, and FRET efficiency, E (Fig. 1B)
Summary
This restriction prohibits monitoring an essential feature of biological processes. While single distances can be read out with high spatio-temporal-resolution, it remains challenging to simultaneously observe conformational changes in different protein parts, map these structural changes as either a result of protein binding or due to intrinsic dynamics[44] and observe how multi-subunit proteins coordinate conformational changes between different domains. PIFE has been initially used in ensemble experiments[20,21,22,23,24,25,26], its use following Cy3 fluorescence at the single molecule level (smPIFE) allowed the identification different protein-interaction modes using immobilized molecules[17,18,27,28,29,30,31,32]. We combine two fluorescence-related effects into one powerful assay that we dub ALEX-based PIFE-FRET It allows observing changes in biochemical structure and interactions by following two distances with two different distance dynamic ranges. To successfully construct and use a two-dimensional ruler thereby introducing PIFE for solution-based single-molecule experiments, we provide a framework for data analysis routine to allow simultaneous and quantitative read-out of two different photophysical parameters: donor brightness (PIFE) and energy transfer efficiency (FRET). We outline possible applications of PIFE-FRET both in studies with diffusing and immobilized molecules indicating the full potential of the technique for mechanistic investigations of biomolecular interactions
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