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Exploring the functional residues in a flavin-binding fluorescent protein using deep mutational scanning.

HyeonSeok Shin¹, Yoobok Cho¹, Dong-hui Choe¹

¹Department of Biological Sciences and KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejon, Republic of Korea.

|June 3, 2014

Summary

Deep mutational scanning identified key amino acid residues in Escherichia coli flavin mononucleotide (FMN)-based fluorescent proteins (EcFbFP). This advance enables the engineering of improved fluorescent protein variants for cellular process monitoring.

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Area of Science:

Biochemistry
Molecular Biology
Protein Engineering

Background:

Flavin mononucleotide (FMN)-based fluorescent proteins are valuable reporters for cellular processes under various conditions.
Limited understanding of individual amino acid roles hinders the development of enhanced fluorescent protein variants.

Purpose of the Study:

To identify functionally important amino acid residues in Escherichia coli flavin mononucleotide binding fluorescent protein (EcFbFP).
To provide insights for engineering improved FMN-based fluorescent proteins.

Main Methods:

Deep mutational scanning coupled with high-throughput sequencing was employed to analyze EcFbFP variants.
Mutational enrichment analysis identified functionally critical residues within the FMN-binding pocket, loops, and near salt bridges.

Main Results:

329 function-retained and 259 function-loss mutations were classified.
Key residues were located in the FMN-binding pocket, protein turns/loops, and near E56-K97 salt bridges.
Site-directed mutagenesis confirmed the mutational sensitivity of identified critical residues.

Conclusions:

Deep mutational scanning is effective for characterizing functional residues in EcFbFP.
Understanding these residues facilitates the rational design of superior FMN-based fluorescent proteins for biotechnological applications.