Monomeric Scarlet Red Fluorescent Protein Variants for Faster Maturation
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Solution Overview
Problem
Monomeric Scarlet Red Fluorescent Protein (mScarlet RFP) has low maturation efficiency, affecting overall cellular brightness, despite having higher intrinsic brightness and quantum yield, making it less suitable for biological imaging.
Innovation Solution
Introduce specific amino acid substitutions such as V196I and G220A, optionally combined with T74I, E219V, T107S, G156V, Y84W, and Y194F, to enhance maturation efficiency and intrinsic brightness, resulting in improved variants like mScarlet3 and mScarlet-I3.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If mScarlet RFP is used, then intrinsic brightness and quantum yield are improved, but maturation efficiency deteriorates
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid residues at specific positions (196, 220, 74, 219, 107, 156, 84, 194) to optimize the balance between intrinsic brightness and maturation efficiency. Through site-directed mutagenesis, the patent generates variants such as V196I, G220A, and their combinations, which alter the protein's maturation kinetics and photophysical properties to achieve both high brightness and efficient maturation.
2Speed
If amino acid substitution T74I is introduced to accelerate maturation, then maturation speed is improved, but intrinsic brightness deteriorates
Solution Approach 1:
The patent merges multiple amino acid substitutions to combine the benefits of accelerated maturation with maintained or improved intrinsic brightness. By combining T74I with other substitutions such as V196I, G220A, and E219V, the patent creates composite variants that achieve both fast maturation kinetics and high phot brightness, resolving the trade-off between speed and intensity.
3Productivity
If multiple amino acid substitutions are introduced to enhance maturation, then maturation efficiency is improved, but protein stability may deteriorate
Solution Approach 1:
The patent applies local quality by introducing amino acid substitutions at specific, strategically chosen positions within the protein structure rather than throughout the entire sequence. The substitutions at positions 196, 220, 74, 219, 107, 156, 84, and 194 are selected to locally optimize maturation efficiency while preserving the overall structural integrity and stability of the fluorescent protein through careful selection of conservative or structurally compatible amino acid replacements.
Data Source
AI summary
A monomeric Scarlet Red Fluorescent protein (mScarlet RFP) includes at least amino acid substitutions V196I and/or G220A when compared to the amino acid sequence of synthetically designed mScarlet RFP. The V196I and/or G220A variants may include one or more further amino acid substitutions.