Followed by the mutation analysis based on the high-throughput sequencing. Although the phenotypes were simply classified into FR and FL subgroups, the relationship between 4000 EcFbFP mutants to their phenotypes was determined in a single run of highthroughput sequencing. Using the depth of the enriched mutations in the FR and FL libraries, we characterized the mutations that are sensitive and tolerant to the loss of EcFbFP function. These data were used to calculate the positional effects of mutations on EcFbFP function. Evaluation of the highly sensitive positions among the FMNbinding identified all the residues that display direct bond formation with the FMN cofactor. However, for hydrophobic interaction of FMN with amino acid residues in the EcFbFP active site, six residues were defined as highly tolerant to mutation. For example, in position L82, the function retained mutation of L82M was highly enriched, while average mutation count in a single mutation was 26.5 for FR library. One possible reason is that the docking of FMN to EcFbFP is not highly specific. FMN binds to the LOV domain of YtvA as a chromophore and becomes covalently linked to the conserved cysteine residue on blue-light activation, thereby activating the signaling cascade of stress s response in B. subtilis. The structure of the LOV domain in YtvA, thus, contains a strong potential for conformational changes upon activation of the photocycle. However, in EcFbFP, only the LOV domain of YtvA is isolated and covalent bond forming cysteine residue, Cys62 is substituted for alanine, which resulted in photo-cycle inhibition. Thus, FMN cofactor is non-covalently bound to the pocket of EcFbFP, and the STAS domain is not linked to the Ja helix. The dynamic potential of EcFbFP for conformational change indicates that the FMN-binding pocket of EcFbFP is not completely rigid and spatially compact. The homo-dimerization of YtvA-LOV domain is mediated by the hydrophobic residues located at Ab,B b,H b, and Ib, and our mutational analysis of FR library agrees with the hydrophobic interaction of homo-dimerization of EcFbFP. Among the 25 highly sensitive positions of EcFbFP, only a single position, V120 located at the Ib sheet, was found to be highly sensitive. Although we speculated that the homo-dimerization is achieved by a cooperative interaction of hydrophobic residues, our analysis suggests that Ib sheet may have a higher sensitivity toward homo-dimer stabilization. The added weight of Ib sheet in homo-dimerization is also supported by the fact that Hb-Ib hairpin moves toward the dimer interface upon light absorption. In addition to residues located at the FMN-binding pocket and hydrophobic homo-dimerization sites, highly sensitive residues were located at loops and turns. Conformational change in YtvA-LOV is initiated from the FMN-binding pocket and leads to the movement of Ea and Ja helices in a scissor-like motion.