Principles of Resonance Energy Transfer

Szöllősi, János; Damjanovich, Sándor; Nagy, Péter; Vereb, György; Mátyus, László

doi:10.1002/0471142956.cy0112s38

Cited by 8 publications

(1 citation statement)

References 76 publications

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“…However, this may not be true for genetically-encoded fluorescent proteins used in FRET studies for several reasons: the fluorophore barrel is large and has a rotation correlation time of about 20-30 ns whereas the FLT is in a range of 1-4 ns (Vogel et al, 2012); the fluorophores are attached to the proteins of interest with a flexible linker (George and Heringa, 2002;Chen et al, 2013Chen et al, , 2013van Rosmalen et al, 2017). A linker length of 15 amino acids is assumed to allow free rotation of the fluorophore (Szöllosi et al, 2006;Chen et al, 2013;Shrestha et al, 2015;van Rosmalen et al, 2017;Ujlaky-Nagy et al, 2018). Even if the assumption is not entirely met, the introduced error is a systematic shift for all samples.…”

Section: Introductionmentioning

confidence: 99%

Three-fluorophore FRET enables the analysis of ternary protein association in living plant cells

Glöckner

Oven-Krockhaus

Rohr

et al. 2019

Preprint

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Integration of signalling on the cellular level is essential for the survival of organisms.Protein-protein interaction studies provide valuable insights in these signalling events.One of the best understood signalling pathways in plants is the brassinosteroid (BR) hormone signalling pathway, which is mediated by the receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) with its co-receptor BRI1-ASSOCIATED KINASE (BAK1). Both BRI1 and BAK1 have been shown to interact with RECEPTOR LIKE PROTEIN 44 (RLP44), which was implicated in cell wall integrity sensing by modulation of BL signalling. Here we provide evidence by quantitative in vivo three-fluorophore FRET-FLIM measurements, that RLP44, BRI1 and BAK1 form a trimeric complex in the plasma membrane of N. benthamiana leaf cells, with an estimated distance between them below 15 nm. The immune receptor FLAGELLIN SENSING 2 (FLS2), which is also a receptor-like kinase like BRI1, is not integrated in a similar complex with RLP44 and BAK1. Our study supports that BRI1 and FLS2 are localized in distinct nanodomains in the PM. As the fluorescence lifetime of the donor is monitored, our method circumvents the extensive calculations necessitated by intensity-based FRET interaction assays and thus provides a feasible base for studying the sub-compartmentalization in the plasma membrane of living plant cells with a nanoscale resolution.

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Section: Introductionmentioning

confidence: 99%

Three-fluorophore FRET enables the analysis of ternary protein association in living plant cells

Glöckner

Oven-Krockhaus

Rohr

et al. 2019

Preprint

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Heterotrimeric G protein signaling via GIV/Girdin: Breaking the rules of engagement, space, and time

et al. 2016

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Canonical signal transduction via heterotrimeric G proteins is spatially and temporally restricted, i.e., triggered exclusively at the plasma membrane (PM), only by agonist activation of G-protein-coupled receptors (GPCRs) via a process that completes within a few hundred milliseconds. Recently, a rapidly emerging paradigm has revealed a non-canonical pathway for activation of trimeric G proteins by the non-receptor guanidine-nucleotide exchange factor (GEF), GIV/Girdin. This pathway has distinctive temporal and spatial features and an unusual profile of receptor engagement: Diverse classes of receptors, not just GPCRs can engage with GIV-GEF to trigger such activation. Such activation is spatially and temporally unrestricted, i.e., can occur both at the PM and on internal membranes discontinuous with the PM, and can continue for prolonged periods of time. Here we review the molecular mechanisms that govern non-canonical G protein activation by GIV-GEF and the relevance of this new paradigm in health and disease.

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Correlative Förster Resonance Electron Transfer‐Proximity Ligation Assay (FRET‐PLA) Technique for Studying Interactions Involving Membrane Proteins

2016

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This unit provides a guide and detailed protocol for studying membrane protein-protein interactions (PPI) using the acceptor-sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of FRET-PLA samples and the detection of correlative FRET/PLA signals as well as on the analysis of FRET-PLA data and interpretation of correlative results when using cyan fluorescent protein (CFP) as a FRET donor and yellow fluorescent protein (YFP) as a FRET acceptor. The correlative application of FRET and PLA combines two powerful tools for monitoring PPI, yielding results that are more reliable than with either technique alone. © 2016 by John Wiley & Sons, Inc.

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Principles of Resonance Energy Transfer

Abstract: This unit describes the basic principles of the fluorescence resonance energy (FRET) process. In addition, it characterizes available parameters and instruments for FRET measurements, discusses limitations, and shows a few examples of the application of FRET.

Cited by 8 publications

References 76 publications

Three-fluorophore FRET enables the analysis of ternary protein association in living plant cells

Three-fluorophore FRET enables the analysis of ternary protein association in living plant cells

Heterotrimeric G protein signaling via GIV/Girdin: Breaking the rules of engagement, space, and time

Correlative Förster Resonance Electron Transfer‐Proximity Ligation Assay (FRET‐PLA) Technique for Studying Interactions Involving Membrane Proteins

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