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Biol. Bull. 201: 231. (October 2001)
© 2001 Marine Biological Laboratory

Introduction to the Featured Report

Green Fluorescent Protein: Enhanced Optical Signals from Native Crystals

"The bioluminescent jellyfish Aequorea emits ‘green’ light in vivo, whereas the pure photoprotein aequorin extracted from the same organism emits ‘blue’ light on addition of Ca2+." Osamu Shimomura made this observation and identified a green fluorescing molecule in 1962; then reported its purification and characterization in 1974 from 30,000 specimens of the hydrozoan jellyfish. The result was green fluorescent protein (GFP), which emits at about 509 nm when it is excited by the blue light (about 460 nm) emitted by aequorin (also purified and characterized by Shimomura). In the jellyfish, this process—called fluorescence resonance energy transfer (FRET)—results in a signal that, because of its longer wavelength, can penetrate farther through the turbidity of natural seawater to its target, which might be, for example, planktonic prey.

The molecular details of GFP emerged about 20 years later (1996) from a pair of independent studies. The laboratories of Roger Tsien and George Phillips, Jr., showed the protein to be an unusual, very regular, barrel-shaped molecule, with its walls (a sheet comprising 11 ß-strands) and caps at both ends of the barrel enclosing and protecting a fluorophore composed of post-translationally modified amino acids.

The gene encoding GFP was cloned by Douglas Prasher and associates in 1992. And shortly thereafter (1994), Martin Chalfie and his laboratory showed that the protein, with its fluorophore, could be completely expressed in bacteria, which would (as if they were jellyfish) glow green when excited with blue light. In the same year, Tulle Hazelrigg demonstrated that a suitable gene construct would express a fusion protein including GFP, and that the site of expression could be precisely located in the organism (Drosophila in this case), or in a single cell, merely by illumination with blue light. With that critical finding, GFP was quickly recognized, and widely used in developmental, cell, neural, and molecular biology, as a reporter of gene expression and a marker for gene product localization.

Recently, Osamu Shimomura asked Shinya Inoué to produce a photomicrograph of the fluorescence emitted by the needle-shaped crystals of purified, native GFP. Inoué agreed, but thought to examine, as well, the anisotropic properties of the crystals. The novel and surprising results of that investigation are set out in the following short report by Inoué and Makoto Goda. In brief, the fluorescence from excited GFP crystals is polarized, with the resonance vectors oriented parallel to the long axis of the crystals. Moreover, when the excitation is also polarized, the fluorescence measured with an analyzer parallel to the crystal is very much higher (by 20–30 times!) than that measured perpendicular to it.

These observations, combined with structural studies involving X-ray crystallography, should shed more light on GFP function and help us improve our interpretation of FRET imaging. Moreover they suggest that, in investigations where dynamic changes in the orientation of GFP-linked motor or contractile proteins are being followed, the use of polarized light might well increase the sensitivity of the observations.

—The Editors

August 2001





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