Abstract
FRET (Fluorescence Resonance Energy Transfer) measurements are commonly applied to proof protein-protein interactions. However, standard methods of live cell FRET microscopy and signal normalization only allow a principle assessment of mutual binding and are unable to deduce quantitative information of the interaction. We present an evaluation and normalization procedure for 3-filter FRET measurements, which reflects the process of complex formation by plotting FRET-saturation curves. The advantage of this approach relative to traditional signal normalizations is demonstrated by mathematical simulations. Thereby, we also identify the contribution of critical parameters such as the total amount of donor and acceptor molecules and their molar ratio. When combined with a fitting procedure, this normalization facilitates the extraction of key properties of protein complexes such as the interaction stoichiometry or the apparent affinity of the binding partners. Finally, the feasibility of our method is verified by investigating three exemplary protein complexes. Altogether, our approach offers a novel method for a quantitative analysis of protein interactions by 3-filter FRET microscopy, as well as flow cytometry. To facilitate the application of this method, we created macros and routines for the programs ImageJ, R and MS-Excel, which we make publicly available.
Highlights
FRET (Förster- or Fluorescence Resonance Energy Transfer) describes a process of radiation-less energy transfer based on dipole-dipole-interactions that can occur from an excited fluorescent molecule to a suitable acceptor molecule[1] (Fig. 1a)
We demonstrate an easy and standardized method to normalize results obtained from 3-filter based FRET experiments in living cells, show how to obtain additional information by the plotting of a FRET saturation curve, and extract biophysical properties of intermolecular interactions by a fitting algorithm
In contrast to NFRET and FRETN, the DFRET value does not suffer from a decline with increasing acceptor concentrations (Fig. 1e) and perfectly follows the saturation characteristics that are typical for a binding reaction according to the law of mass action
Summary
FRET (Förster- or Fluorescence Resonance Energy Transfer) describes a process of radiation-less energy transfer based on dipole-dipole-interactions that can occur from an excited fluorescent molecule (donor) to a suitable acceptor molecule (acceptor)[1] (Fig. 1a). One widely used intensity-based FRET method employs the photodestruction of the acceptor molecules (acceptor bleaching) by strong illumination[10,11] This increases emission of donor fluorescence by eliminating energy dissipation via FRET, but long acquisition times and the destructive nature of the measurement render this method hardly applicable to complex experiments in living cells or set-ups other than microscopy. The experimental design of this method is comparatively easy and, while mostly used in microscopy, can be applied to many different set-ups, including high-throughput methods such as slide-15,16 and flow-based cytometry[17,18,19] These methods allow the assessment of FRET in entire cell populations, which highly increases the statistical power and permits a more precise extraction of underlying variables from a dataset. While specialized equipment allows the use of fluorescence lifetime measurements in flow cytometry[20,21], intensity based methods are far more common[22,23]
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