title: Mispacking and the Fitness Landscape of the Green Fluorescent Protein Chromophore Milieu.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Shounak
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Christian D
authors.initials: CD
authors.lastname: Schenkelberg
authors.affiliation: Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Jordan
authors.affiliation: Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Reimertz
authors.affiliation: Department of Biology, Colgate University , Hamilton, New York 13346, United States.
authors.forename: Emily E
authors.initials: EE
authors.lastname: Crone
authors.affiliation: Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Donna E
authors.initials: DE
authors.lastname: Crone
authors.affiliation: Department of Biological Sciences, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.|Department of Computer Science, Rensselaer Polytechnic Institute , Troy, New York 12180, United States.
authors.forename: Christopher
authors.identifier: 0000-0002-2813-3716
authors.initials: C
authors.lastname: Bystroff
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005453:Fluorescence; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D037402:Hydrozoa; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins