Cyan Fluorescent Protein Carries a Constitutive Mutation That Prevents Its Dimerization

• Published in: Biochemistry (Easton) 2011-02, Vol.50 (4), p.437-439
• Main Authors: Espagne, Agathe, Erard, Marie, Madiona, Karine, Derrien, Valérie, Jonasson, Gabriella, Lévy, Bernard, Pasquier, Hélène, Melki, Ronald, Mérola, Fabienne
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution; Amino Acid Substitution - genetics; Biochemistry, Molecular Biology; Chemical Sciences; Dimerization; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Fluorescence Resonance Energy Transfer - methods; Fluorescent Dyes; Fluorescent Dyes - chemistry; Genetic Variation; Green Fluorescent Proteins; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Life Sciences; Models, Molecular; Mutation; or physical chemistry; Protein Binding; Protein Multimerization; Protein Multimerization - genetics; Recombinant Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Theoretical and; Ultracentrifugation
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi1015875

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.