期刊
CELLS
卷 9, 期 11, 页码 -出版社
MDPI
DOI: 10.3390/cells9112381
关键词
FRET; live-cell measurements; flow cytometry; peroxisomes; PEX5; peroxisomal targeting signal
类别
资金
- Austrian Science Fund [SFB-F54, P-27842]
- Agency for Science, Technology and Research (A*STAR) of Singapore
- Austrian Science Fund FWF [I2738-B21]
Measuring Forster-resonance-energy-transfer (FRET) efficiency allows the investigation of protein-protein interactions (PPI), but extracting quantitative measures of affinity necessitates highly advanced technical equipment or isolated proteins. We demonstrate the validity of a recently suggested novel approach to quantitatively analyze FRET-based experiments in living mammalian cells using standard equipment using the interaction between different type-1 peroxisomal targeting signals (PTS1) and their soluble receptor peroxin 5 (PEX5) as a model system. Large data sets were obtained by flow cytometry coupled FRET measurements of cells expressing PTS1-tagged EGFP together with mCherry fused to the PTS1-binding domain of PEX5, and were subjected to a fitting algorithm extracting a quantitative measure of the interaction strength. This measure correlates with results obtained by in vitro techniques and a two-hybrid assay, but is unaffected by the distance between the fluorophores. Moreover, we introduce a live cell competition assay based on this approach, capable of depicting dose- and affinity-dependent modulation of the PPI. Using this system, we demonstrate the relevance of a sequence element next to the core tripeptide in PTS1 motifs for the interaction strength between PTS1 and PEX5, which is supported by a structure-based computational prediction of the binding energy indicating a direct involvement of this sequence in the interaction.
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