title: Exploring the functional residues in a flavin-binding fluorescent protein using deep mutational scanning.
abstract: Flavin mononucleotide (FMN)-based fluorescent proteins are versatile reporters that can monitor various cellular processes in both aerobic and anaerobic conditions. However, the understanding of the role of individual amino acid residues on the protein function has been limited and has restricted the development of better functional variants. Here we examine the functional amino acid residues of Escherichia coli flavin mononucleotide binding fluorescent protein (EcFbFP) using the application of high-throughput sequencing of functional variants, termed deep mutational scanning. The variants were classified into 329 function-retained (FR) and 259 function-loss (FL) mutations, and further the mutational enrichment in each amino acid residues was weighed to find the functionally important residues of EcFbFP. We show that the crucial amino acid residues of EcFbFP lie among the FMN-binding pocket, turns and loops of the protein where conformation changes occur, and spatially clustered residues near the E56-K97 salt bridges. In addition, the mutational sensitivity of the critical residues was confirmed by site-directed mutagenesis. The deep mutational scanning of EcFbFP has demonstrated important implications for constructing better functioning protein variants.
authors.affiliation: Department of Biological Sciences and KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejon, Republic of Korea.
authors.forename: HyeonSeok
authors.initials: H
authors.lastname: Shin
authors.affiliation: Department of Biological Sciences and KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejon, Republic of Korea.
authors.forename: Yoobok
authors.initials: Y
authors.lastname: Cho
authors.affiliation: Department of Biological Sciences and KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejon, Republic of Korea.
authors.forename: Dong-hui
authors.initials: DH
authors.lastname: Choe
authors.affiliation: Department of Biological Sciences and KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejon, Republic of Korea.
authors.forename: Yujin
authors.initials: Y
authors.lastname: Jeong
authors.affiliation: Department of Biological Sciences and KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejon, Republic of Korea.
authors.forename: Suhyung
authors.initials: S
authors.lastname: Cho
authors.affiliation: Department of Biological Sciences and KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejon, Republic of Korea; Intelligent Synthetic Biology Center, Daejon, Republic of Korea.
authors.forename: Sun Chang
authors.initials: SC
authors.lastname: Kim
authors.affiliation: Department of Biological Sciences and KAIST Institute for the BioCentury, Korea Advanced Institute of Science and Technology, Daejon, Republic of Korea; Intelligent Synthetic Biology Center, Daejon, Republic of Korea.
authors.forename: Byung-Kwan
authors.initials: BK
authors.lastname: Cho
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D005486:Flavin Mononucleotide / Q000378:metabolism; D005415:Flavins / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008164:Luminescent Proteins / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D059645:Mutation Rate; D011485:Protein Binding; D055503:Protein Multimerization
chemical_list: D000596:Amino Acids; D029968:Escherichia coli Proteins; D005415:Flavins; D008164:Luminescent Proteins; D050505:Mutant Proteins; D005486:Flavin Mononucleotide