The biochemical properties of a protein are coded by its amino acid sequence. However, the fitness landscape of individual amino acids that contribute to the florescence of EcFbFP are unknown, except for certain functional residues such as the conserved cysteine and glutamine. Thus, the usage of FMN as a chromophore facilitates EcFbFP to be a promising alternative class of reporter protein capable of expression in both aerobic and anaerobic systems. Mutation of the cysteine residue to alanine (designated as EcFbFP) increases the fluorescence by tenfold compared to the wild-type LOV domain. In the subsequent reaction where energy is reverted to the ground state, conformational change occurs, which allows emission at the wavelength of 495 nm, wherein the FMN cofactor is non-covalently buried in the LOV domain. In the LOV domain, a metastable covalent bond between a conserved cysteine residue (Cys62) and atom C(4a) of the FMN ring is formed by blue light irradiation (450 nm). Among these FbFPs, Escherichia coli FbFP (EcFbFP) which consists of 137 amino acids was engineered from the photoactive LOV (light-oxygen-voltage) domain of Bacillus subtilis YtvA, which is a 261-amino acid protein consisting of two functional domains, a core LOV domain and a sulfate transporter and anti-sigma factor (STAS) domain connected by a long linker. Bacterial FbFPs respond to light blue and activate stress-related signaling pathways. Alternatively, the flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) can be employed as an in vivo reporter system –. However, GFP-based fluorescent proteins require molecular oxygen for the generation of cyclic tripeptide chromophore, which has been a major drawback in the GFP-based reporter protein applications under conditions with limited oxygen supply. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist.įluorescent reporter proteins derived from the green fluorescent protein (GFP) and its derivatives have been used to analyze a wide array of cellular processes such as gene regulation, protein localization, and protein interaction. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.įunding: The authors acknowledge financial support from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031957, 2011-0031962) and the Basic Core Technology Development Program for the Oceans and the Polar Regions (2011-0021053) funded by the Ministry of Education, Science and Technology. Received: JanuAccepted: ApPublished: June 2, 2014Ĭopyright: © 2014 Shin et al. Gill, University of Edinburgh, United Kingdom (2014) Exploring the Functional Residues in a Flavin-Binding Fluorescent Protein Using Deep Mutational Scanning. The deep mutational scanning of EcFbFP has demonstrated important implications for constructing better functioning protein variants.Ĭitation: Shin H, Cho Y, Choe D-h, Jeong Y, Cho S, Kim SC, et al. In addition, the mutational sensitivity of the critical residues was confirmed by site-directed mutagenesis. 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. 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. 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. 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. Flavin mononucleotide (FMN)-based fluorescent proteins are versatile reporters that can monitor various cellular processes in both aerobic and anaerobic conditions.
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