Photoconvertible Fluorescent Proteins With Higher Contrast and Less Blinking
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Solution Overview
Problem
Current photoconvertible fluorescent proteins, such as mEos4b, suffer from poor photoconversion contrast and high photoblinking, limiting their utility in quantitative applications like single molecule photoactivated localization microscopy.
Innovation Solution
Engineered photoconvertible fluorescent proteins, such as Janus and Ignis, with specific mutations at residues 41, 70, and optionally 197 (e.g., Met41Ile, Val70Thr, and Ile197Met) to enhance photoconversion contrast and reduce photoblinking.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If mEos4b is used as a photoconvertible fluorescent protein, then fixation resistance is achieved, but photoconversion contrast is poor and photoblinking is high
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues (M41I, V70T, I197M) to alter the chromophore's pKa values and photochemical properties. This changes the physical-chemical parameters of the fluorescent protein to achieve both high photoconversion contrast and reduced photoblinking while maintaining fixation resistance.
Solution Approach 2:
The patent applies local quality by introducing specific mutations at targeted positions (residues 41, 70, and 197) rather than throughout the entire protein. These localized changes specifically affect the chromophore environment and photoconversion properties without compromising the overall structural stability and fixation resistance of the protein.
2Reliability
If mEos4b is used as a photoconvertible fluorescent protein, then fixation resistance is achieved, but photoblinking rate is high
Solution Approach 1:
The patent changes the photochemical parameters of the fluorescent protein through targeted mutations, which modify the chromophore's electronic structure and reduce non-radiative transitions that cause photoblinking, thereby decreasing the photoblinking rate while preserving fixation resistance.
Solution Approach 2:
The patent introduces localized mutations at specific positions (41, 70, 197) that specifically address the photoblinking issue by modifying the chromophore environment, without affecting the overall protein structure and fixation resistance properties.
3Measurement precision
If photoconversion efficiency is increased through mutations, then red state brightness is improved, but protein structure stability may be affected
Solution Approach 1:
The patent carefully selects mutations that change the chromophore environment parameters (pKa, hydrogen bonding, steric constraints) to improve photoconversion efficiency and red state brightness, while the mutations are positioned to minimize disruption to the overall protein fold and structural stability.
Solution Approach 2:
The patent applies local quality by introducing mutations only at specific positions (41, 70, 197) that are strategically located to influence chromophore photochemistry without compromising the global structural integrity of the protein, thus improving photoconversion while maintaining stability.
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 engineered proteins exhibit improved photoconversion rates and reduced photoblinking, enabling better imaging precision and accuracy in single molecule localization microscopy.
Implementation Method 1
genetically-encoded photoconvertible fluorescent proteins are important tools for single molecule photoactivated localization microscopy
Implementation Method 2
photoconvertible fluorescent proteins comprising one or more mutations or substitutions of the mEos4b protein coding sequence
Data Source
AI summary
Disclosed herein, are photoconvertible fluorescent proteins or analogs thereof, and in particular, green-to-red photoconvertible fluorescent proteins or analogs thereof of the EosFP family; and compositions comprising the same and methods for analyzing a physiologically active substance in a cell wherein the fluorescent proteins are expressed in the cell.