Through the flexibility of Ea-Fa loops and Da-Ea loops by the glycine residues at Gly67, Gly59, and Gly26, which support the importance of flexibility in the most dynamically moving structures, EF loops, Ea helix, and Ja helix. On the contrary, residues Tyr52, Gly51, Lys97, and Gly99 spatially clustered near the GH hairpin and CD loops suggest the importance of the structure stabilization role of the salt bridge between K97 and E56. This data further supports the highly conserved salt-bridge in PAS domains, and the only function retaining mutations at these residues are K97R and E56D, which are capable of forming arginine-glutamate salt bridge and lysine-aspartate salt bridge, respectively. We speculated that this salt bridge must be conserved to stabilize the structure in functional form. This notion is further supported by the fact that the salt bridge plays a structure-stabilizing role rather than a role in the receptor activation of the phot1 LOV2 domain in A. thaliana. Our method differs from the general screening of positive phenotype by random mutagenesis, as the negative phenotype is the main target. However, our deep mutational scanning data also indicates 329 unique mutations that preserve the function along with the enrichment of each mutation. Although the variant selection method was based on the presence or absence of function, the analytical approach can be more useful if applied to other mutation induction methods coupled with functional assays. The diverse range of functional variants can be sub-categorized by increase, decrease, retention, and complete loss of function. Consequently, enriched residues for different functional subcategories can be analyzed using our analytical method that allows comparison of different and conserved enrichment locations between subcategories. Thus, allowing functional weight determination of amino acid residues enables more detailed analysis of protein sequence and function relationship. Another application is the functional analysis of co-existing mutations. The synthetic functional rescue library is a library screened for function gain from loss of function mutants. This method coupled with multiple barcoded adaptors for each cycles, allow the analysis of co-existing mutations similar to the synthetic lethality concept. We suspect that this analysis of multiple mutant behaviors adds another dimension to the investigation of protein function. Our study determined that the most sensitive residues of EcFbFP lies among the bond-formation residues of the FMNbinding pocket, dynamic conformational changing turns and loops, and cluster of residues near the E56-K97 salt-bridge formation sites. These results indicate that similar methodology can be applied to find functionally important residues in a wide array of enzymes. Deep mutational scanning was focused on the functionally sensitive residues to find residues that play important roles in EcFbFP as well as the LOV domain.