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. 2002 Apr 2;99(7):4272-7.
doi: 10.1073/pnas.062065199.

Fluorescence Polarization of Green Fluorescence Protein

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Free PMC article

Fluorescence Polarization of Green Fluorescence Protein

Shinya Inoué et al. Proc Natl Acad Sci U S A. .
Free PMC article

Erratum in

  • Proc Natl Acad Sci U S A 2002 Jun 25;99(13):9081

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.

Figures

Figure 1
Figure 1
(A and B) Fluorescence of GFP crystals illuminated with <460-nm wavelength plane polarized light and observed through 527 ± 15-nm barrier filter. The transmission axis of the polarizer is oriented horizontally in A and vertically in B. No analyzer was present. (Bar = 30 μm.)
Figure 2
Figure 2
Normalized fluorescence intensities of crystal versus orientation angle of microscope stage. (A) Open circles: intensity measurements with polarizer present, analyzer absent. Polarizer transmission axis is at 0°. Solid line: plot of I/I0 = 14.05 × cos2φ + 2.65 (from Eq. 1). (B) Open circles: intensity measurements with analyzer present but no polarizer. Analyzer transmission axis is at 0°. Solid line: Plot of I/I0 = 14.55 × cos2φ + 2.40 (from Eq. 2). (C) Open circles: Intensity measurements with both polarizer and analyzer present with their axes parallel to each other and oriented at 0°. Solid line: plot of I/I0 = 22.5 × cos4φ + 2.8 × cos2φ + 1 (from Eq. 3). (D) Open circles: intensity measurements with both polarizer and analyzer present with their axes crossed. Solid line: plot of I/I0 = −11.25 × cos4φ + 11.0 × cos2φ + 2.15 (from Eq. 4). The data points in each graph are for an individual crystal with the microscope stage turned every 10°. The points are shown in measured sequence from left to right, with their maxima normalized to 1.0. Except in B and D, the graphs were derived from separate crystals. The intensities in B and D were both normalized by using the peak value in B.
Figure 3
Figure 3
(A and B) Dichroism of GFP crystals observed with quartz halogen illuminator. Double-headed arrows: polarizer transmission axis. (Bar = 30 μm.) (C) Confocal epifluorescence optical sections taken 4 μm above, at, and 4 μm below the mid plane of GFP crystal with large hollow core. The asymmetry of the image above and below the midplane and the strong flare reflects the high refractive index of the crystal wall material. (Bar = 10 μm.)
Figure 4
Figure 4
Schematic of crystal structure. (A) Orientations relative to the long crystal axis (cc′) of: major dipole for fluorophores (ff′), slow birefringence axis (ss′), and β can major axis (bb′). (B) Published structure of GFP molecule based on x-ray crystallographic analyses (6, 7). (C) Schematic array of molecules fitting conditions shown in A and B. While maintaining the parallel alignment of the chromophore ellipsoids' major axes, this schematic could be modified with the backbone of the β cans rotated around the long crystal axis to occupy two or more discrete orientations.

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