Multimerizable Protein Fusion for Intracellular Interaction Detection
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Solution Overview
Problem
Conventional methods for determining protein-protein interactions in cells lack the ability to provide positional and temporal information on these interactions, especially in an intracellular environment, and are often cumbersome due to requirements for fluorescent protein optimization and potential interference from cross-excitation and bleed-through.
Innovation Solution
The use of fusion proteins comprising a protein of interest and a multimerizable protein, along with a fluorescent protein, which form assemblies upon interaction, allowing for the detection of fluorescent foci to determine protein-protein interactions and their duration within cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods (surface plasmon resonance, protein mass spectroscopy, anisotropy measurements) are used to determine protein-protein interactions, then interaction detection is possible, but positional and temporal information on the interactions cannot be obtained
Solution Approach 1:
The invention introduces a multimerizable protein as an intermediary component that forms visible assemblies when target proteins interact. This mediator enables the detection of protein-protein interactions while preserving spatial and temporal information through the formation of discrete fluorescent foci that can be imaged and tracked over time
Solution Approach 2:
The invention utilizes fluorescent proteins that emit visible light signals when multimerizable proteins form assemblies upon target protein interaction. The fluorescent signal provides both positional information (location of foci) and temporal information (timing and duration of interactions) without disrupting the natural interaction
2Loss of information
If fluorescent proteins are used to determine protein-protein interactions in living cells, then positional and temporal information can be obtained, but the method becomes complicated due to linker optimization requirements and cross-excitation issues
Solution Approach 1:
The invention extracts the fluorescent reporting function from a complex FRET system and places it on a separate multimerizable protein component. This separation eliminates the need for donor-acceptor pairs and linker optimization, while still providing fluorescent signal for detecting protein interactions
Solution Approach 2:
The invention uses multiple copies of the multimerizable protein domain (e.g., PB1 domains) that self-assemble into visible complexes. This copying approach creates amplifiable signals that are easy to detect without requiring complex optical setups or multiple fluorescent protein types
3Illumination intensity
If reconstituted fluorescent proteins are used to determine protein-protein interactions, then fluorescence emission occurs, but the period until interaction ends and interaction duration cannot be determined because fluorescent proteins do not dissociate
Solution Approach 1:
The invention employs a dynamic multimerizable protein system that can reversibly assemble and disassemble based on target protein interaction status. When target proteins interact, multimerizable proteins form visible assemblies; when interactions cease, the assemblies dissociate. This dynamic behavior enables real-time tracking of interaction duration and reversibility
Solution Approach 2:
The multimerizable protein assemblies can be discarded (disassembled) when target protein interactions end, and recovered (reassembled) when interactions resume. This reversible process allows continuous monitoring of interaction dynamics, including duration and frequency, without permanent fluorescent labeling
4Measurement precision
If proteins are forcibly translocated to particular sites in cells for interaction determination, then interaction detection is possible, but natural intracellular environment and positional information are lost
Solution Approach 1:
The multimerizable proteins attached to target proteins self-assemble into visible complexes at the natural location where target proteins interact. The system serves itself by using the target proteins' natural localization and interaction patterns to guide assembly formation, eliminating the need for artificial translocation or confinement to specific cellular compartments
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
Enables the precise determination of protein-protein interactions in their natural intracellular environment, providing both positional and temporal information, and improves the efficiency of homomultimer formation detection compared to existing methods.
Implementation Method 1
a first fusion protein comprising the first protein, a multimerizable protein, and a fluorescent protein
Data Source
AI summary
A method for determining an interaction between a first protein and a second protein comprises the steps of:expressing in a cell or introducing into a cella first fusion protein comprising the first protein, a multimerizable protein, and a fluorescent protein, anda second fusion protein comprising the second protein and a multimerizable protein;detecting a fluorescent focus formed by an association between the first fusion protein and the second fusion protein in the cell; anddetermining an interaction between the first protein and the second protein according to the detection of the fluorescent focus.