Rational Structure-Based Design of Bright GFP-Based Complexes with Tunable Dimerization

Autor: Thorsten Wohland, Chiew Ling Lim, Yuemin Celina Chee, Andreas Grüter, Gregor Jung, Ralf Jauch, Guangyu Sun, Swaine L. Chen, Majid Eshaghi
Rok vydání: 2015
Předmět:
Zdroj: Angewandte Chemie International Edition. 54:13952-13956
ISSN: 1433-7851
DOI: 10.1002/anie.201506686
Popis: 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.
Databáze: OpenAIRE