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

Angew Chem Int Ed Engl. 2015 Nov 16;54(47):13952-6. doi: 10.1002/anie.201506686. Epub 2015 Oct 8.


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.

Keywords: dimerization; fluorescent probes; green fluorescent protein; protein engineering; single-molecule studies.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antibodies / chemistry*
  • Drug Design*
  • Escherichia coli / chemistry
  • Escherichia coli / cytology
  • Fluorescent Antibody Technique
  • Green Fluorescent Proteins / chemistry*
  • HeLa Cells
  • Humans
  • Hydrogen-Ion Concentration
  • Models, Molecular
  • Protein Conformation
  • Protein Multimerization*


  • Antibodies
  • Green Fluorescent Proteins