Proteins from Anthozoa species are homologous to the green fluorescent protein (GFP) from Aequorea victoria but with absorption/emission properties extended to longer wavelengths. HcRed is a far-red fluorescent protein originating from the sea anemone Heteractis crispa with absorption and emission maxima at 590 and 650nm, respectively. We use ultrasensitive fluorescence spectroscopic methods to demonstrate that HcRed occurs as a dimer in solution and to explore the interaction between chromophores within such a dimer. We show that red chromophores within a dimer interact through a Förster-type fluorescence resonance energy transfer (FRET) mechanism. We present spectroscopic evidence for the presence of a yellow chromophore, an immature form of HcRed. This yellow chromophore is involved in directional FRET with the red chromophore when both types of chromophores are part of one dimer. We show that by combining ensemble and single molecule methods in the investigation of HcRed, we are able to sort out subpopulations of chromophores with different photophysical properties and to understand the mechanism of interaction between such chromophores. This study will help in future quantitative microscopy investigations that use HcRed as a fluorescent marker.
Bibliographical noteFunding Information:
We acknowledge Dr. K. A. Lukyanov from the Institute of Bioorganic Chemistry in Moscow for supplying the HcRed and Dr. G. S. Waldo from the Los Alamos National Laboratory, New Mexico, for supplying the super folder GFP used in these studies. We thank Dr. Richard Keller from the Los Alamos National Laboratory, New Mexico, for helpful comments and suggestions. This work was supported by the Laboratory-Directed Research and Development Program of the Los Alamos National Laboratory, New Mexico; the Research Council of Katholieke Universiteit Leuven, Belgium (GOA2006/2); and the Flemish Science Foundation, Belgium.
- fluorescence microscopy
- fluorescence resonance energy transfer
- green fluorescent proteins
- single-molecule methods
ASJC Scopus subject areas
- Electronic, Optical and Magnetic Materials
- Atomic and Molecular Physics, and Optics
- Biomedical Engineering