title: Local fitness landscape of the green fluorescent protein.
abstract: Fitness landscapes depict how genotypes manifest at the phenotypic level and form the basis of our understanding of many areas of biology, yet their properties remain elusive. Previous studies have analysed specific genes, often using their function as a proxy for fitness, experimentally assessing the effect on function of single mutations and their combinations in a specific sequence or in different sequences. However, systematic high-throughput studies of the local fitness landscape of an entire protein have not yet been reported. Here we visualize an extensive region of the local fitness landscape of the green fluorescent protein from Aequorea victoria (avGFP) by measuring the native function (fluorescence) of tens of thousands of derivative genotypes of avGFP. We show that the fitness landscape of avGFP is narrow, with 3/4 of the derivatives with a single mutation showing reduced fluorescence and half of the derivatives with four mutations being completely non-fluorescent. The narrowness is enhanced by epistasis, which was detected in up to 30% of genotypes with multiple mutations and mostly occurred through the cumulative effect of slightly deleterious mutations causing a threshold-like decrease in protein stability and a concomitant loss of fluorescence. A model of orthologous sequence divergence spanning hundreds of millions of years predicted the extent of epistasis in our data, indicating congruence between the fitness landscape properties at the local and global scales. The characterization of the local fitness landscape of avGFP has important implications for several fields including molecular evolution, population genetics and protein design.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.|Nizhny Novgorod State Medical Academy, Minin Sq. 10/1, 603005 Nizhny Novgorod, Russia.|Central European Institute of Technology, Masaryk University, Brno 62500, Czech Republic.|Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, 88 Dr. Aiguader, 08003 Barcelona, Spain.|Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.
authors.forename: Karen S
authors.initials: KS
authors.lastname: Sarkisyan
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.|Central European Institute of Technology, Masaryk University, Brno 62500, Czech Republic.
authors.forename: Dmitry A
authors.initials: DA
authors.lastname: Bolotin
authors.affiliation: Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, 88 Dr. Aiguader, 08003 Barcelona, Spain.|Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.
authors.forename: Margarita V
authors.initials: MV
authors.lastname: Meer
authors.affiliation: Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, 88 Dr. Aiguader, 08003 Barcelona, Spain.|Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.|Moscow Institute of Physics and Technology, Institutskiy Pereulok 9, g.Dolgoprudny 141700, Russia.
authors.forename: Dinara R
authors.initials: DR
authors.lastname: Usmanova
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.|Nizhny Novgorod State Medical Academy, Minin Sq. 10/1, 603005 Nizhny Novgorod, Russia.
authors.forename: Alexander S
authors.initials: AS
authors.lastname: Mishin
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.|Faculty of Medicine, Moscow State University, Lomonosov Avenue 31/5, Moscow 119192, Russia.
authors.forename: George V
authors.initials: GV
authors.lastname: Sharonov
authors.affiliation: Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, 88 Dr. Aiguader, 08003 Barcelona, Spain.|Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.|Laboratory of Protein Physics, Institute of Protein Research of the Russian Academy of Sciences, 4 Institutskaya Str., Pushchino, Moscow Region 142290, Russia.
authors.forename: Dmitry N
authors.initials: DN
authors.lastname: Ivankov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
authors.forename: Nina G
authors.initials: NG
authors.lastname: Bozhanova
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.|Pirogov Russian National Research Medical University, Ostrovitianov 1, Moscow 117997, Russia.
authors.forename: Mikhail S
authors.initials: MS
authors.lastname: Baranov
authors.affiliation: Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, 88 Dr. Aiguader, 08003 Barcelona, Spain.|Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.
authors.forename: Onuralp
authors.initials: O
authors.lastname: Soylemez
authors.affiliation: Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, 88 Dr. Aiguader, 08003 Barcelona, Spain.|Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.|Laboratory of Protein Physics, Institute of Protein Research of the Russian Academy of Sciences, 4 Institutskaya Str., Pushchino, Moscow Region 142290, Russia.
authors.forename: Natalya S
authors.initials: NS
authors.lastname: Bogatyreva
authors.affiliation: Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, 88 Dr. Aiguader, 08003 Barcelona, Spain.|Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.
authors.forename: Peter K
authors.initials: PK
authors.lastname: Vlasov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.
authors.forename: Evgeny S
authors.initials: ES
authors.lastname: Egorov
authors.affiliation: Pirogov Russian National Research Medical University, Ostrovitianov 1, Moscow 117997, Russia.|A. A. Kharkevich Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow 127051, Russia.|Department of Bioinformatics and Bioengineering, Moscow State University, Moscow 119234, Russia.
authors.forename: Maria D
authors.initials: MD
authors.lastname: Logacheva
authors.affiliation: Department of Bioinformatics and Bioengineering, Moscow State University, Moscow 119234, Russia.|Department of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.
authors.forename: Alexey S
authors.initials: AS
authors.lastname: Kondrashov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.|Central European Institute of Technology, Masaryk University, Brno 62500, Czech Republic.
authors.forename: Dmitry M
authors.initials: DM
authors.lastname: Chudakov
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.|Central European Institute of Technology, Masaryk University, Brno 62500, Czech Republic.
authors.forename: Ekaterina V
authors.initials: EV
authors.lastname: Putintseva
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.|Central European Institute of Technology, Masaryk University, Brno 62500, Czech Republic.
authors.forename: Ilgar Z
authors.initials: IZ
authors.lastname: Mamedov
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
authors.forename: Dan S
authors.initials: DS
authors.lastname: Tawfik
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, 117997 Moscow, Russia.|Nizhny Novgorod State Medical Academy, Minin Sq. 10/1, 603005 Nizhny Novgorod, Russia.
authors.forename: Konstantin A
authors.initials: KA
authors.lastname: Lukyanov
authors.affiliation: Bioinformatics and Genomics Programme, Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, 88 Dr. Aiguader, 08003 Barcelona, Spain.|Universitat Pompeu Fabra (UPF), 08003 Barcelona, Spain.|Institució Catalana de Recerca i Estudis Avançats (ICREA), 23 Pg. Lluís Companys, 08010 Barcelona, Spain.
authors.forename: Fyodor A
authors.initials: FA
authors.lastname: Kondrashov
mesh_terms: D000818:Animals; D004843:Epistasis, Genetic; D019143:Evolution, Molecular; D005453:Fluorescence; D056726:Genetic Association Studies; D056084:Genetic Fitness; D005838:Genotype; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism*; D037402:Hydrozoa / Q000737:chemistry / Q000235:genetics; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010641:Phenotype
chemical_list: D050505:Mutant Proteins; D049452:Green Fluorescent Proteins