Mispacking and the Fitness Landscape of the Green Fluorescent Protein Chromophore Milieu.


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.

Submission Details

ID: B7Nwc2uR

Submitter: Connie Wang

Submission Date: July 31, 2017, 11:46 a.m.

Version: 2

Publication Details
Banerjee S;Schenkelberg CD;Jordan TB;Reimertz JM;Crone EE;Crone DE;Bystroff C,Biochemistry (2017) Mispacking and the Fitness Landscape of the Green Fluorescent Protein Chromophore Milieu. PMID:28074648
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1S6Z 2004-01-28T00:00:00+0000 1.5 Enhanced Green Fluorescent Protein Containing the Y66L Substitution
6LNP 2019-12-31T00:00:00+0000 2.99 Crystal structure of citrate Biosensor
2QZ0 2007-08-15T00:00:00+0000 1.2 Mature Q183E variant of Green Fluorescent Protein Chromophore
6MB2 2018-08-29T00:00:00+0000 5.0 Cryo-EM structure of the PYD filament of AIM2
1W7S 2004-09-09T00:00:00+0000 1.85 Wild-Type Aequorea victoria Green Fluorescent Protein
3O77 2010-07-30T00:00:00+0000 2.35 The structure of Ca2+ Sensor (Case-16)
7CD8 2020-06-18T00:00:00+0000 2.0 GFP-40/GFPuv complex, Form II
7CD7 2020-06-18T00:00:00+0000 1.7 GFP-40/GFPuv complex, Form I
6ZUI 2020-07-23T00:00:00+0000 2.2 Crystal structure of the Cys-Ser mutant of the cpYFP-based biosensor for hypochlorous acid
2Q6P 2007-06-05T00:00:00+0000 2.1 The Chemical Control of Protein Folding: Engineering a Superfolder Green Fluorescent Protein

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
95.0 Green fluorescent protein (mutated to super folder GFP, see PDB) P42212 GFP_AEQVI