title: Rational Structure-Based Design of Bright GFP-Based Complexes with Tunable Dimerization.
abstract: Fluorescent proteins are transformative tools; thus, any brightness increase is a welcome improvement. We invented the "vGFP strategy" based on structural analysis of GFP bound to a single-domain antibody, predicting tunable dimerization, enhanced brightness (ca. 50%), and improved pH resistance. We verified all of these predictions using biochemistry, crystallography, and single-molecule studies. We applied the vsfGFP proteins in three diverse scenarios: single-step immunofluorescence in vitro (3× brighter due to dimerization); expression in bacteria and human cells in vivo (1.5× brighter); and protein fusions showing better pH resistance in human cells in vivo. The vGFP strategy thus allows upgrading of existing applications, is applicable to other fluorescent proteins, and suggests a method for tuning dimerization of arbitrary proteins and optimizing protein properties in general.
authors.affiliation: National University of Singapore, Department of Medicine, Yong Loo Lin School of Medicine, 1E Kent Ridge Road, NUHS Tower Block, Level 10, Singapore 119074 (Singapore).
authors.forename: Majid
authors.initials: M
authors.lastname: Eshaghi
authors.affiliation: National University of Singapore, Departments of Chemistry and Biological Sciences and Center for Bio-Imaging Sciences, Singapore 117557 (Singapore).
authors.forename: Guangyu
authors.initials: G
authors.lastname: Sun
authors.affiliation: Saarland University, 66123 Saarbrücken (Germany).
authors.forename: Andreas
authors.initials: A
authors.lastname: Grüter
authors.affiliation: National University of Singapore, Department of Medicine, Yong Loo Lin School of Medicine, 1E Kent Ridge Road, NUHS Tower Block, Level 10, Singapore 119074 (Singapore).
authors.forename: Chiew Ling
authors.initials: CL
authors.lastname: Lim
authors.affiliation: National University of Singapore, Department of Medicine, Yong Loo Lin School of Medicine, 1E Kent Ridge Road, NUHS Tower Block, Level 10, Singapore 119074 (Singapore).
authors.forename: Yuemin Celina
authors.initials: YC
authors.lastname: Chee
authors.affiliation: Saarland University, 66123 Saarbrücken (Germany).
authors.forename: Gregor
authors.initials: G
authors.lastname: Jung
authors.affiliation: Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530 (China).
authors.forename: Ralf
authors.initials: R
authors.lastname: Jauch
authors.affiliation: National University of Singapore, Departments of Chemistry and Biological Sciences and Center for Bio-Imaging Sciences, Singapore 117557 (Singapore).
authors.forename: Thorsten
authors.initials: T
authors.lastname: Wohland
authors.affiliation: National University of Singapore, Department of Medicine, Yong Loo Lin School of Medicine, 1E Kent Ridge Road, NUHS Tower Block, Level 10, Singapore 119074 (Singapore). slchen@gis.a-star.edu.sg.|Genome Institute of Singapore, Infectious Diseases Group, 60 Biopolis Street, Genome, #02-01, Singapore 138672 (Singapore). slchen@gis.a-star.edu.sg.
authors.forename: Swaine L
authors.initials: SL
authors.lastname: Chen
mesh_terms: D000906:Antibodies / Q000737:chemistry*; D015195:Drug Design; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology; D005455:Fluorescent Antibody Technique; D049452:Green Fluorescent Proteins / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011487:Protein Conformation; D055503:Protein Multimerization
chemical_list: D000906:Antibodies; D049452:Green Fluorescent Proteins
keywords: dimerization; fluorescent probes; green fluorescent protein; protein engineering; single-molecule studies