Single-Chain Photoswitch Protein for Light-Controlled Split Protein Binding
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Solution Overview
Problem
Conventional photoswitching proteins, such as Magnets, have limitations in binding efficiency and require high intracellular concentrations, and there is a need for improved control of protein binding and dissociation.
Innovation Solution
A one-molecule-type photoswitching protein (scMagnet) is developed by linking pMag and nMag in series, utilizing their structural changes under light to efficiently control the binding and dissociation of split proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a two-molecule-type Magnet system (pMag and nMag) is used to control protein binding and dissociation, then the system shows improved control efficiency compared to wild-type VVD, but the binding efficiency depends on intracellular concentration and interaction frequency is limited
Solution Approach 1:
The patent merges the two separate magnet proteins (pMag and nMag) into a single fused photoswitching protein by linking them through a peptide linker. This single-molecule system eliminates the need for two independent molecules to interact, thereby dramatically improving binding efficiency and interaction frequency while maintaining light-controlled dimerization capability
Solution Approach 2:
The fused photoswitching protein is segmented into distinct functional domains (pMag and nMag) connected by a peptide linker, allowing each domain to retain its photoswitching properties while enabling intramolecular interaction. This segmentation within a single molecule resolves the dependency on intermolecular concentration and interaction frequency
2Adaptability or versatility
If VVD forms a homodimer upon blue light irradiation, then the protein can be used for photoswitching applications, but it has low binding affinity and cannot select binding partners
Solution Approach 1:
The patent introduces local charge differences by substituting specific amino acids in the dimerization interface of VVD with basic or acidic amino acids. This creates localized positive (pMag) or negative (nMag) charges that enhance electrostatic attraction and binding affinity while maintaining the overall photoswitching functionality of the VVD core structure
Solution Approach 2:
The patent creates asymmetric magnet proteins by introducing different amino acid substitutions in the dimerization interface of different VVD molecules. This asymmetry enables selective binding between complementary magnets (positive and negative) while preventing homodimerization, thereby improving binding affinity and partner selectivity
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 scMagnet system enhances the efficiency of protein binding and dissociation by 10 times or more compared to two-molecule systems, with minimal leak activity, and can control various proteins like split Cas9 endonuclease, Cre recombinase, and Flp recombinase in a light-dependent manner.
Implementation Method 1
Vivid (VVD), a protein derived from Neurospora crassa, receives a blue light and rapidly forms a homodimer
Implementation Method 2
a VVD in which at least one of the amino acids located in the contact region during dimer formation has been substituted with a basic amino acid (an amino acid having a positive charge)
Data Source
Figure 1a~2d
Figure 3a~4
Figure 5~7d
AI summary
It is an object of the present invention to provide a photoswitching protein that can control the binding and dissociation of proteins more efficiently than conventional photoswitching proteins. It is a photoswitching protein consisting of a single polypeptide, differing from the conventional photoswitching protein consisting of two different proteins. More specifically, the photoswitching protein of the present invention is, for example, a protein in which the C-terminus of the following protein (a) is directly or indirectly linked to the N-terminus of the following protein (b): (a) a protein comprising an amino acid sequence having a sequence identity of 80% or more to an amino acid sequence comprising a deletion of an X number of consecutive amino acid residues from the C-terminal residue to the N-terminal side of the amino acid sequence as set forth in SEQ ID No: 1 or SEQ ID No: 36, wherein X represents an integer of 0 or greater and 6 or smaller; and (b) a protein comprising an amino acid sequence having a sequence identity of 80% or more to an amino acid sequence comprising a deletion of a Y number of consecutive amino acid residues from the N-terminal residue to the C-terminal side of the amino acid sequence as set forth in SEQ ID No: 1 or SEQ ID No: 36, wherein Y represents an integer of 0 or greater and 55 or smaller.