Cyan fluorescent protein carries a constitutive mutation that prevents its dimerization

Biochemistry. 2011 Feb 1;50(4):437-9. doi: 10.1021/bi1015875. Epub 2010 Dec 30.

Abstract

The tendency of GFP-like fluorescent proteins to dimerize in vitro is a permanent concern as it may lead to artifacts in FRET imaging applications. However, we have found recently that CFP and YFP (the couple of GFP variants mostly used in FRET studies) show no trace of association in the cytosol of living cells up to millimolar concentrations. In this study, we investigated the oligomerization properties of purified CFP, by fluorescence anisotropy and sedimentation velocity. Surprisingly, we found that CFP has a much weaker homoaffinity than other fluorescent proteins (K(d) ≥ 3 × 10(-3) M), and that this is due to the constitutive N146I mutation, originally introduced into CFP to improve its brightness.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution / genetics
  • Dimerization
  • Fluorescence Polarization
  • Fluorescence Resonance Energy Transfer / methods
  • Fluorescent Dyes / chemistry*
  • Genetic Variation
  • Green Fluorescent Proteins / chemistry*
  • Green Fluorescent Proteins / genetics*
  • Models, Molecular
  • Mutation*
  • Protein Binding
  • Protein Multimerization / genetics*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Ultracentrifugation

Substances

  • Cyan Fluorescent Protein
  • Fluorescent Dyes
  • Recombinant Proteins
  • Green Fluorescent Proteins