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.
Highlights
Fluorescent 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 [1,2]
Bacterial FbFPs respond to light blue and activate stress-related signaling pathways [6]. Among these FbFPs, Escherichia coli FbFP (EcFbFP) which consists of 137 amino acids was engineered from the photoactive LOV 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 [7]
The pEcFbFP was transformed into E. coli TOP10, which were grown on Luria broth (LB) agar plates containing 50 mg/mL ampicillin and 1 mM isopropyl-b-d-thiogalactopyranoside (IPTG)
Summary
Fluorescent 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 [1,2]. The flavin mononucleotide (FMN)-based fluorescent proteins (FbFPs) can be employed as an in vivo reporter system [3,4,5]. Bacterial FbFPs respond to light blue and activate stress-related signaling pathways [6]. 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 [7]. 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). 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 [7,8]
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