Fluorescent protein barrel fluctuations and oxygen diffusion pathways in mCherry.

Area of Science:

• Biochemistry
• Biophysics
• Molecular Biology

Background:

• Fluorescent proteins (FPs) are essential biochemical markers for cellular process studies.
• Red fluorescent proteins (RFPs) are ideal for in vivo applications due to low autofluorescence in the red spectrum.
• Monomeric RFPs, like mFruits, offer advantages over naturally occurring dimeric/tetrameric forms.

Purpose of the Study:

• Investigate oxygen diffusion pathways within the mCherry protein.
• Understand the relationship between protein barrel fluctuations and oxygen pathways.
• Identify strategies to improve FP photostability and chromophore maturation.

Main Methods:

• Utilized molecular dynamics calculations to simulate mCherry protein dynamics.
• Employed implicit ligand sampling to map oxygen entry points to the chromophore.
• Analyzed the impact of strategic amino acid substitutions on protein fluctuations and oxygen pathways.

Main Results:

• Identified specific oxygen diffusion pathways from the solvent into the mCherry chromophore.
• Demonstrated that protein barrel fluctuations influence oxygen accessibility.
• Showed that amino acid substitutions can effectively block or modify these pathways and reduce fluctuations.

Conclusions:

• Understanding oxygen pathways is crucial for optimizing FP photostability and maturation.
• Protein engineering via targeted amino acid substitutions can enhance mCherry's properties.
• This study provides insights for developing more robust RFPs for advanced biological imaging.