Probing dimerization and intraprotein fluorescence resonance energy transfer in a far-red fluorescent protein from the sea anemone Heteractis crispa.
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 650 nm, 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 Forster-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.
Lessard
Guillaume A
GA
Los Alamos National Laboratory, Mail Stop J567, Los Alamos, New Mexico 87545, USA.
Habuchi
Satoshi
S
Werner
James H
JH
Goodwin
Peter M
PM
De Schryver
Frans
F
Hofkens
Johan
J
Cotlet
Mircea
M
D000818:Animals; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008164:Luminescent Proteins / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods*; D015336:Molecular Probe Techniques; D012615:Sea Anemones / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein