Anomalous negative fluorescence anisotropy in yellow fluorescent protein (YFP 10C): quantitative analysis of FRET in YFP dimers

Biochemistry. 2007 Dec 18;46(50):14403-17. doi: 10.1021/bi701575n. Epub 2007 Nov 21.

Abstract

Yellow fluorescent protein (YFP) is widely used as a genetically encoded fluorescent marker in biology. In the course of a comprehensive study of this protein, we observed an unusual, negative fluorescence anisotropy at pH 6.0 (McAnaney, T. B., Zeng, W., Doe, C. F. E., Bhanji, N., Wakelin, S., Pearson, D. S., Abbyad, P., Shi, X., Boxer, S. G., and Bagshaw, C. R. (2005) Biochemistry 44, 5510-5524). Here we report that the fluorescence anisotropy of YFP 10C depends on protein concentration in the low micromolar range that was not expected. We propose that the negative anisotropy is a result of unidirectional Förster resonance energy transfer (FRET) in a dimer of YFP, with the donor chromophore in the neutral form and the acceptor chromophore in the anionic form. This unusual mechanism is supported by studies of a monomeric YFP (A206K YFP) and transient-absorption spectroscopy of YFP 10C. A detailed analysis of the chromophore transition dipole moment direction is presented. The anisotropy and rate constant of this energy transfer are consistent with values produced by an analysis of the dimer structure observed in crystals.

Publication types

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

MeSH terms

  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Dimerization
  • Fluorescence Polarization / methods*
  • Fluorescence Resonance Energy Transfer / methods*
  • Luminescent Proteins / chemistry*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism

Substances

  • Bacterial Proteins
  • Luminescent Proteins
  • Recombinant Proteins
  • yellow fluorescent protein, Bacteria