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Fluorescence polarization of green fluorescence protein.

ABSTRACT: We report here the striking anisotropy of fluorescence exhibited by crystals of native green fluorescence protein (GFP). The crystals were generated by water dialysis of highly purified GFP obtained from the jellyfish Aequorea. We find that the fluorescence becomes six times brighter when the excitation, or emission, beam is polarized parallel (compared with perpendicular) to the crystal long axis. Thus, the major dipoles of the fluorophores must be oriented very nearly parallel to the crystal long axis. Observed in a polarizing microscope between parallel polars instead of either a polarizer or analyzer alone, the fluorescence polarization ratio rises to an unexpectedly high value of about 30:1, nearly the product of the fluorescence excitation and emission ratios, suggesting a sensitive method for measuring fluorophore orientations, even of a single fluorophore molecule. We have derived equations that accurately describe the relative fluorescence intensities of crystals oriented in various directions, with the polarizer and analyzer arranged in different configurations. The equations yield relative absorption and fluorescence coefficients for the four transition dipoles involved. Finally, we propose a model in which the elongated crystal is made of GFP molecules that are tilted 60 degrees to align the fluorophores parallel to the crystal long axis. The unit layer in the model may well correspond to the arrangement of functional GFP molecules, to which resonant energy is efficiently transmitted from Ca2+-activated aequorin, in the jellyfish photophores.

SUBMITTER: Inoue S

PROVIDER: S-EPMC123638 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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Fluorescence polarization of green fluorescence protein.

Inoué Shinya S Shimomura Osamu O Goda Makoto M Shribak Mykhailo M Tran P T PT

Proceedings of the National Academy of Sciences of the United States of America 20020401 7

We report here the striking anisotropy of fluorescence exhibited by crystals of native green fluorescence protein (GFP). The crystals were generated by water dialysis of highly purified GFP obtained from the jellyfish Aequorea. We find that the fluorescence becomes six times brighter when the excitation, or emission, beam is polarized parallel (compared with perpendicular) to the crystal long axis. Thus, the major dipoles of the fluorophores must be oriented very nearly parallel to the crystal l ...[more]

PMID: 11929998

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Project description:UnaG is a recently discovered ligand-induced fluorescent protein that utilizes bound bilirubin (BR) as its fluorophore. The fluorescence of the UnaG-BR complex (holoUnaG) compares in quantum efficiency to that of enhanced green fluorescent protein, but it is superior in that the fluorophore formation is instantaneous and not dependent on oxygen; hence, much attention has been paid to UnaG as a new fluorescent probe. However, many important molecular properties of fluorescent probes remain unknown, such as the association/dissociation rates of BR, which determine the stability thereof, and the dispersibility of UnaG in aqueous solutions, which influence the functions of labeled proteins. In this study, we found, in the process of investigating the association rate, that the holoUnaG takes two distinct fluorescence states, which we named holoUnaG1 and holoUnaG2. The holoUnaG1 initially forms after binding BR and then changes to the brighter holoUnaG2 by a reversible intra-molecular reaction, thereby finally reaching an equilibrium between the two states. Spectroscopic analysis indicated that the intra-molecular reaction was associated not with a chemical change of BR but with a change in the environmental conditions surrounding BR. We also revealed that the molecular brightness ratio and equilibrium population ratio of the two states (holoUnaG1/holoUnaG2) were 1:3.9 and 6:4, respectively, using photon number counting analysis. From these results, we have suggested a novel schema, to our knowledge, for the formation of the UnaG and BR complex system and have determined the various rate constants associated therein. Additionally, using analytical ultracentrifugation, we established that UnaG in the apo-state (apoUnaG) and the holoUnaG are monomeric in aqueous solution. These findings provide not only key information for the practical use of UnaG as a fluorescent probe, but also the possibility for development of a brighter UnaG mutant by genetic engineering to constitutive holoUnaG2.

Dataset Information

Fluorescence polarization of green fluorescence protein.

Publications

Fluorescence polarization of green fluorescence protein.

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