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SPECTRAL PERTURBATIONS OF THE AEQUOREA GREEN‐FLUORESCENT PROTEIN
Author(s) -
Ward William W.,
Prentice Hugh J.,
Roth Amy F.,
Cody Chris W.,
Reeves Sue C.
Publication year - 1982
Publication title -
photochemistry and photobiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.818
H-Index - 131
eISSN - 1751-1097
pISSN - 0031-8655
DOI - 10.1111/j.1751-1097.1982.tb02651.x
Subject(s) - chromophore , absorbance , ionic strength , aequorea victoria , green fluorescent protein , fluorescence , chemistry , luminescence , circular dichroism , analytical chemistry (journal) , isoelectric point , photochemistry , materials science , chromatography , crystallography , aqueous solution , optics , biochemistry , physics , enzyme , optoelectronics , gene
— In the jellyfish Aequorea, the green‐fluorescent protein (GFP) functions as the in vivo bio‐luminescence emitter via energy transfer from the photoprotein aequorin. Accumulated evidence has indicated that the Aequorea GFP is a relatively inflexible protein. Present evidence, however, indicates that the chromophore environment is readily accessible to a variety of external perturbants. Native Aequorea GFP has an absorbance maximum at 395 nm and a shoulder at 470 nm. In low ionic strength buffer at neutral pH and room temperature the 395/470 nm absorbance ratio is about 2.0. We show that this ratio is highly variable depending upon temperature, ionic strength, protein concentration, and pH. A maximum ratio of 6.5 (at a protein concentration of 18.6 mg/m/) and minimum of 0.42 (at a pH of 12.2) have been measured. In the latter case, the resulting absorption and excitation spectra resemble those of Renilla GFP in spectral shape (but not wavelength maximum). In all cases as the perturbant is varied the resulting spectra pass through a sharp isosbestic point, suggesting a relatively simple two‐state mechanism. These spectral perturbations are fully reversible. On the basis of these results, we suggest that the chromophore binding site is conformationally flexible. pH‐Dependent changes in the near‐UV and visible circular dichroism spectra plus spectrophotometric titration of tyrosine residues lend additional support to this hypothesis.