Estimating the Interaction Strength Between PTS1-Peptides and Their Receptor PEX5 in Living Cells Using Flow-Cytometer-Based FRET (flowFRET) Measurements.

Autor:	Hochreiter B; Institute for Vascular Biology and Thrombosis Research, Medical University of Vienna, Vienna, Austria. bernhard.hochreiter@meduniwien.ac.at., Schmid JA; Institute for Vascular Biology and Thrombosis Research, Medical University of Vienna, Vienna, Austria., Berger J; Department of Pathobiology of the Nervous System, Medical University of Vienna, Vienna, Austria., Kunze M; Department of Pathobiology of the Nervous System, Medical University of Vienna, Vienna, Austria. markus.kunze@meduniwien.ac.at.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2023; Vol. 2643, pp. 413-434.
DOI:	10.1007/978-1-0716-3048-8_30
Abstrakt:	The import of many peroxisomal matrix proteins is initiated by the interaction of type-1 peroxisomal targeting signals (PTS1) residing at the extreme C-terminus of cargo proteins and their receptor protein PEX5. This interaction has been amply investigated by biophysical methods using isolated proteins and peptides or heterologous systems such as two-hybrid assays. However, a recently developed novel application of Fluorescence resonance energy transfer (FRET) allows a quantifying measurement of this interaction in living cells. This method combines the systematic measurement of FRET-efficiency in a high number of cells with a well-suited normalization protocol and a fitting algorithm, which together allow the estimation of numerical values for the apparent interaction strength that correlates with other measures of binding strength but can be obtained under rather physiological conditions. (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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