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Rational Structure‐Based Design of Bright GFP‐Based Complexes with Tunable Dimerization
Author(s) -
Eshaghi Majid,
Sun Guangyu,
Grüter Andreas,
Lim Chiew Ling,
Chee Yuemin Celina,
Jung Gregor,
Jauch Ralf,
Wohland Thorsten,
Chen Swaine L.
Publication year - 2015
Publication title -
angewandte chemie
Language(s) - English
Resource type - Journals
eISSN - 1521-3757
pISSN - 0044-8249
DOI - 10.1002/ange.201506686
Subject(s) - green fluorescent protein , fluorescence , in vivo , chemistry , rational design , protein tag , biophysics , immunofluorescence , in vitro , protein design , molecule , brightness , protein structure , biochemistry , combinatorial chemistry , nanotechnology , recombinant dna , antibody , materials science , biology , fusion protein , gene , physics , microbiology and biotechnology , organic chemistry , optics , quantum mechanics , immunology
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.