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Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein
Published on: June 18, 2020
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First biphotochromic fluorescent protein moxSAASoti stabilized for oxidizing environment
N K Marynich1, M G Khrenova1,2, A V Gavshina1
1A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia.
Scientific Reports
|May 13, 2022
Summary
This study engineered a novel biphotochromic protein variant, moxSAASoti, with enhanced photoswitching and photoconversion rates. The F177 side chain
Area of Science:
- Biochemistry
- Photochemistry
- Protein Engineering
Background:
- Biphotochromic proteins exhibit reversible photoswitching and irreversible photoconversion.
- Cysteine residue reactivity necessitates development of oxidation-resistant proteins like "mox" variants.
- Previous studies focused on monomeric and oxidation-resistant protein designs.
Purpose of the Study:
- To engineer a novel moxSAASoti variant with improved photoswitching and photoconversion.
- To investigate the role of F177 side chain conformational flexibility in protein function.
- To establish a correlation between protein pKa values and chromophore structure.
Main Methods:
- Site-saturated mutagenesis at C105 and C117 to create the moxSAASoti variant (C21N/C71G/C105G/C117T/C175A).
- Classical Molecular Dynamics (MD) simulations to analyze F177 side chain conformational behavior.
- Quantum Mechanics/Molecular Mechanics (QM/MM) simulations to correlate pKa values with chromophore C-O bond length.
Main Results:
- The moxSAASoti variant demonstrated higher on-to-off photoswitching rates, more complete off-to-on recovery, and increased photoconversion rates compared to mSAASoti.
- F177 side chain flexibility was identified as the primary determinant of off-to-on conversion rates.
- A direct relationship was found between the C-O bond length of the neutral chromophore's phenyl fragment and the protein's pKa value.
Conclusions:
- The engineered moxSAASoti variant offers superior performance for biphotochromic applications.
- F177 side chain dynamics provide a measurable indicator for conversion rates.
- The C-O bond length serves as a predictive tool for red fluorescent protein pKa values.
