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- Title
Rational Structure-Based Design of Bright GFP-Based Complexes with Tunable Dimerization.
- Authors
Eshaghi, Majid; Sun, Guangyu; Grüter, Andreas; Lim, Chiew Ling; Chee, Yuemin Celina; Jung, Gregor; Jauch, Ralf; Wohland, Thorsten; Chen, Swaine L.
- 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 (3x brighter due to dimerization); expression in bacteria and human cells in vivo (1.5x 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.
- Subjects
GREEN fluorescent protein; DIMERIZATION; IMMUNOFLUORESCENCE; SINGLE molecules; PROTEIN engineering; FLUORESCENT probes
- Publication
Angewandte Chemie, 2015, Vol 127, Issue 47, p14158
- ISSN
0044-8249
- Publication type
Article
- DOI
10.1002/ange.201506686