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Circular permutation of ligand-binding module improves dynamic range of genetically encoded FRET-based nanosensor
Satoshi Okada1, Kazuhisa Ota, Takashi Ito
1Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8561, Japan.
Protein Science : a Publication of the Protein Society
|October 15, 2009
Summary
This study enhances fluorescent indicator protein (FLIP) nanosensors by engineering bacterial periplasmic binding proteins (PBPs). Circular permutation significantly increases their dynamic range for more reliable small molecule quantification in biological systems.
Area of Science:
- Biochemistry
- Molecular Biology
- Biophysics
Background:
- Quantitative measurement of small molecules is crucial for understanding metabolic regulation.
- Fluorescent indicator protein (FLIP) nanosensors utilize bacterial periplasmic binding proteins (PBPs) and Förster resonance energy transfer (FRET) for sensing.
- Enhancing the dynamic range of FLIP nanosensors is key to improving measurement reliability.
Purpose of the Study:
- To expand the dynamic range of FLIP nanosensors.
- To improve the performance of PBP-based FLIP nanosensors through protein engineering.
- To broaden the applicability of FLIP technology for metabolite quantification.
Main Methods:
- Engineered bacterial periplasmic binding proteins (PBPs) using circular permutation and hinge loop deletion.
- Constructed FLIP nanosensors incorporating modified PBPs and Förster resonance energy transfer (FRET) between cyan and yellow variants of green fluorescent protein (GFP).
- Validated nanosensor performance through in vitro FRET measurements and in vivo applications in complex solutions and living yeast cells.
Main Results:
- Six circularly permutated PBPs demonstrated significantly larger dynamic ranges compared to their native forms.
- Circular permutation enabled three previously non-functional PBPs to serve as effective ligand-binding modules in FLIP nanosensors.
- Successfully quantified amino acid concentrations and measured amino acid influx in real-time within living yeast cells.
Conclusions:
- Circular permutation is an effective strategy to enhance the dynamic range and functionality of PBP-based FLIP nanosensors.
- This engineering approach improves the performance of existing nanosensors and expands the range of measurable metabolites.
- The enhanced FLIP nanosensor technology offers a powerful tool for precise metabolic analysis in complex biological environments.
