Sequence and function relationship of Escherichia coli flavin mononucleotide binding fluorescent protein

Abstract

Flavin mononucleotide (FMN)-binding fluorescent proteins can provide in vivo reporter system, without oxygen. Here, we present the functional landscape of individual amino acid sequence of Escherichia coli FMN-binding fluorescent protein (EcFbFP). We used random mutagenesis to generate the mutant libraries, which were screened by function loss or retained. The function and sequence relationship of the mutants were analyzed in single high-throughput sequencing, resulting in 329 tolerant mutations and 259 sensitive mutations that show retained fluorescence or loss of fluorescence respectively. In addition, the enrichment of tolerant or sensitive mutations in each amino acid residues were weighed to find functionally important residues n EcFbFP. The mutation enrichment analysis show that the positions critical to the function of EcFbFP lies among the FMN binding pocket, turns and loops of the protein where dynamic conformational changes occur and the Glu56-Lys97 salt bridge which is critical to structural stability of EcFbFP. Collectively, the mutational scanning results provide a functional landscape of each amino acid of EcFbFP.