GPCR Internal Sensors With cpFPs for High-SNR Imaging
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current methods for studying GPCR-drug interactions in living systems suffer from low signal-to-noise ratio (SNR) and dynamic range, limiting their applicability, and existing fluorescent sensors consume a large portion of the optical spectrum, making multiplex imaging and use alongside optogenetic effectors difficult.
Innovation Solution
Development of single-wavelength fluorescent sensors integrated into the third intracellular loop of GPCRs, utilizing circularly permuted fluorescent proteins (cpFPs) with optimized peptide linkers, enhancing sensitivity and dynamic range, allowing for improved visualization and multiplex imaging.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If FRET-based sensors with multiple fluorescent proteins are used to study GPCR-drug interactions, then conformational changes can be detected, but the signal-to-noise ratio and dynamic range are very low
Solution Approach 1:
The patent extracts and eliminates the acceptor fluorescent protein component from the FRET system, using only a single donor fluorescent protein whose emission intensity directly reports GPCR conformational changes. This removal of the acceptor component resolves the technical contradiction by improving signal-to-noise ratio while reducing sensor structural complexity.
Solution Approach 2:
The patent uses a single fluorescent protein that undergoes intrinsic emission changes in response to GPCR conformational changes, rather than relying on energy transfer between multiple proteins. This single-protein copying approach improves measurement precision by eliminating the low signal-to-noise ratio inherent in FRET-based systems.
2Measurement precision
If FRET-based sensors with multiple fluorescent proteins are used to study GPCR-drug interactions, then conformational changes can be detected, but the dynamic range is very low
Solution Approach 1:
The patent removes the acceptor fluorescent protein from the FRET system, utilizing only a single donor fluorescent protein. This extraction simplifies the sensor structure while expanding the dynamic range by eliminating the limitations of energy transfer efficiency and spectral overlap that constrain FRET-based dynamic range.
Solution Approach 2:
The single fluorescent protein copies the conformational information of the GPCR through intrinsic emission intensity changes, providing a broader dynamic range compared to FRET systems. This approach resolves the contradiction by achieving high measurement precision with simplified sensor structure.
3Measurement precision
If multiple fluorescent proteins with partially overlapping excitation/emission spectrum are inserted into GPCR, then conformational changes can be detected, but a large portion of the available spectrum is consumed
Solution Approach 1:
The patent extracts and eliminates the need for multiple fluorescent proteins by using a single donor fluorescent protein whose emission intensity directly reports conformational changes. This resolves the spectral congestion problem, freeing up the optical spectrum for multiplex imaging applications while maintaining conformational detection capability.
Solution Approach 2:
The single fluorescent protein serves multiple functions: detecting GPCR conformational changes and preserving spectral bandwidth for other imaging purposes. This multi-functionality resolves the contradiction by maintaining detection capability while improving adaptability for multiplex imaging.
4Reliability
If FRET-based sensors are used in living systems, then GPCR-drug interactions can be studied, but the low signal-to-noise ratio limits applicability
Solution Approach 1:
The single fluorescent protein copies GPCR conformational information through intrinsic emission changes, providing high signal-to-noise ratio measurements suitable for living systems. This resolves the contradiction by achieving reliable applicability in living systems through improved measurement precision.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The new sensors provide superior sensitivity, broader dynamic range, and faster kinetics, enabling effective visualization of GPCR interactions in living systems and facilitating multiplex imaging and use with optogenetic effectors.
Implementation Method 1
Fluorescent sensors based on circularly permuted single FPs (either green fluorescent protein or related FPs) have been engineered to sense small molecules so that these can be visualized directly with a change in fluorescence intensity of the chromophore
Data Source
AI summary
Provided are circularly permuted fluorescent protein sensors useful to integrate into the third intracellular loop of a G protein-coupled receptor (GPCR). Also provided are GPCRs having a circularly permuted fluorescent protein sensor integrated into its third intracellular loop and methods of using such GPCRs, e.g., to screen for GPCR agonists and antagonists and to monitor activation of GPCRs both in vitro and in vivo.