RT Journal Article
SR Electronic
T1 PIFE meets FRET – a molecular ruler goes 2D
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 047779
DO 10.1101/047779
A1 Evelyn Ploetz
A1 Eitan Lerner
A1 Florence Husada
A1 Martin Roelfs
A1 SangYoon Chung
A1 Johannes Hohlbein
A1 Shimon Weiss
A1 Thorben Cordes
YR 2016
UL http://biorxiv.org/content/early/2016/04/08/047779.abstract
AB 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 gp5/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.