Exploring the Functional Residues in a Flavin-Binding Fluorescent Protein Using Deep Mutational Scanning

<div><p>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 <i>Escherichia coli</i> 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.</p></div

Critical residues determined by mutation frequency.

<p>(a) Common and unique mutations in the FL library and FR library. (b) Heatmap of the positions of unique mutations of the FR and FL library with mutation frequency drawn on a scale of 0 to 1. Positions with enriched mutations are shown in yellow. The positional effect bar shows the residues with enriched FL mutations in yellow and residues with enriched FR mutations in black. The asterisks indicate known FMN-binding sites.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Mutation frequency of amino acid residues involved in hydrophobic interaction with FMN.

<p>Mutation frequency of amino acid residues involved in hydrophobic interaction with FMN.</p

Mapping statistics of FR and FL library.

<p>Mapping statistics of FR and FL library.</p

The relationship of critical residues to EcFbFP function.

<p>(a) Mapping of the frequency of amino acid changes in 25 sensitive residues of EcFbFP. Known or predicted function of each residue is (I) formation of salt bridge with FMN, (II) formation of hydrogen bond with FMN, (III) hydrophobic contact with FMN, (IV) hydrogen bond with FMN’s ribityl chain, (V) FMN-binding sites predicted by MSDsite, (VI) dimer formation site, and (VII) turns and loops. (b) Structural mapping of the critical residues of EcFbFP generated by the PyMOL software (The PyMOL Molecular Graphics System, Version 1.1r1, Schrödinger, LLC) showing residues 20 to 137 of the LOV domain of YtvA (PDB: 2PR6). (i) Critical residues categorized as FMN-binding sites (I, II, III, and VI) shown in green spheres, dimer forming site (V) shown in violet sphere, and turns and loops are (VII) shown in yellow spheres. To highlight the directional interaction of amino acid side chains of each residue, the spheres in the FMN-binding sites and dimer forming sites show the Cβ atom and the spheres in remaining sites show the Cα atom. (ii) The α-helices is shown in red, β-sheets shown in yellow, loops, hairpins and FMN shown in green. The secondary structures are labeled for clarification. (c) Results of the site-directed mutagenesis of the variants (i) <i>in vivo</i> and (ii) in purified form under UV illumination. (iii) The excitation spectrum of purified protein between the wavelength of 360 nm to 470 nm with fixed emission wavelength of 495 nm with fixed emission wavelength of 495 nm, and (iv) <i>in vitro</i> measurement of emission spectrum of purified protein between the wavelength of 480 nm to 550 nm with fixed excitation wavelength of 450 nm.</p