The autocatalytic maturation of the chromophore in green fluorescent protein (GFP) was thought to require the precise positioning of the side chains surrounding it in the core of the protein, many of which are strongly conserved among homologous fluorescent proteins. In this study, we screened for green fluorescence in an exhaustive set of point mutations of seven residues that make up the chromophore microenvironment, excluding R96 and E222 because mutations at these positions have been previously characterized. Contrary to expectations, nearly all amino acids were tolerated at all seven positions. Only four point mutations knocked out fluorescence entirely. However, chromophore maturation was found to be slower and/or fluorescence reduced in several cases. Selected combinations of mutations showed nonadditive effects, including cooperativity and rescue. The results provide guidelines for the computational engineering of GFPs.
Submitter: Connie Wang
Submission Date: July 31, 2017, 11:46 a.m.
|Number of data points||134|
|Proteins||Green fluorescent protein (mutated to super folder GFP, see PDB)|
|Assays/Quantities/Protocols||Experimental Assay: In Vivo Relative Fluorescence|
|Libraries||Single point mutants of sfGFP at 7 positions near the chromophore|
|Percent Identity||Matching Chains||Protein||Accession||Entry Name|
|95.0||Green fluorescent protein (mutated to super folder GFP, see PDB)||P42212||GFP_AEQVI|