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Fluorescent Protein Fluorophore Maturation Mechanisms

Autocatalytic formation of the fluorophore (also referred to as a chromophore) within the shielded environment of the polypeptide backbone during fluorescent protein maturation follows a surprisingly unified mechanism, especially considering the diverse natural origins of these useful biological probes. Shortly after synthesis, most fluorescent proteins slowly mature through a multi-step process that consists of folding, initial fluorophore ring cyclization, and subsequent modifications of the fluorophore. The spectral properties of fluorescent proteins are dependent upon the structure of the fluorophore as well as the localized interactions of amino acid residues in the immediate vicinity, and in some cases, residues far removed from the fluorophore. The interactive tutorials linked below explore fluorophore formation in a wide variety of spectrally diverse fluorescent proteins deduced from crystallographic studies.

Formation of the GFP Fluorophore - Among the most remarkable attributes of the original green fluorescent protein (GFP) derived from the Aequorea victoria jellyfish, as well as the more recently developed palette of color-shifted genetic variants, is that the entire 27 kiloDalton polypeptide structure is essential for the development and maintenance of fluorescence in this very remarkable family of proteins. The principle fluorophore (often termed a chromophore) is a tripeptide consisting of the residues serine, tyrosine, and glycine at positions 65-67 in the sequence. Although this simple amino acid motif is commonly found throughout nature, it does not generally result in fluorescence. This interactive tutorial explores the molecular re-arrangement that occurs during the formation of the enhanced green fluorescent protein (EGFP) fluorophore, which substitutes threonine for serine at position 65 in the amino acid sequence.

Formation of the DsRed Fluorophore - The red fluorescent protein, DsRed, was discovered in the Anthozoan genus Discosoma during a search in reef corals for naturally occurring GFP analogues emitting fluorescence in longer wavelength regions. Although DsRed shares only approximately 26 percent sequence homology with the Aequorea victoria green fluorescent protein, enough critical amino acid motifs are conserved to form a similar very stable three-dimensional beta-can barrel structure. The fluorescence emission spectrum of DsRed is shifted to longer wavelengths by almost 75 nanometers (583-nanometer maximum) compared to the enhanced green fluorescent protein (EGFP). This interactive tutorial explores the molecular re-arrangement that occurs during the formation of the DsRed fluorescent protein fluorophore, which features a similar imidazoline ring system, but substitutes glutamine for serine as the first amino acid residue in the tripeptide sequence.

Formation of the ZsYellow (zFP538) Fluorophore - The yellow fluorescent protein, ZsYellow (originally referred to as zFP538), was discovered in the Anthozoan button polyp Zoanthus. Although ZsYellow shares only approximately 28 percent sequence homology with the original Aequorea victoria green fluorescent protein, enough critical amino acid motifs are conserved to form a similar very stable three-dimensional beta-can barrel structure. One of the most unique features of the ZsYellow fluorescence emission spectrum is that the peak (538 nanometers) occurs almost midway between those of GFP (508 nanometers) and DsRed (583 nanometers), presenting an opportunity to investigate proteins emitting fluorescence in the yellow portion of the visible light spectrum. This interactive tutorial explores the molecular re-arrangement that occurs during the formation of the ZsYellow fluorescent protein fluorophore, which features a novel three-ring system and peptide backbone cleavage due to the substitution of lysine for serine as the first amino acid residue in the chromophore tripeptide sequence.

Formation of the eqFP611 Fluorophore - The far-red fluorescent protein, eqFP611, was isolated from the sea anemone Entacmaea quadricolor and displays one of the largest Stoke's shifts and red-shifted emission wavelength profiles of any naturally occurring Anthozoan fluorescent protein. Although eqFP611 shares only approximately 23 percent sequence homology with the Aequorea victoria green fluorescent protein, enough critical amino acid motifs are conserved to form a very stable three-dimensional beta-can barrel structure, a consistent motif in fluorescent proteins. The fluorescence emission spectrum of eqFP611 is shifted to longer wavelengths by almost 105 nanometers (displaying a maximum at 611-nanometers) when compared to the enhanced green fluorescent protein (EGFP). This interactive tutorial explores the series of molecular re-arrangements that occur during the formation of the eqFP611 fluorescent protein fluorophore, which features a similar imidazoline ring system to EGFP, but substitutes methionine for serine as the first amino acid residue in the tripeptide sequence.

Formation of the HcRed Fluorophore - The far-red fluorescent protein, HcRed, was discovered through site-directed and random mutagenesis efforts on a non-fluorescent chromoprotein (hcCP) isolated from the Indo-Pacific Anthozoa species, Heteractis crispa. Although HcRed shares only approximately 21 percent amino acid sequence homology with the Aequorea victoria green fluorescent protein, enough critical amino acid motifs are conserved to form a very stable three-dimensional beta-can barrel structure. The fluorescence emission spectrum of HcRed is shifted to longer wavelengths by almost 140 nanometers (645-nanometer peak; emitting in the far red) compared to the enhanced green fluorescent protein (EGFP). This interactive tutorial explores the series of molecular re-arrangements that occur during the formation of the HcRed fluorescent protein fluorophore, which features a similar imidazoline ring system to EGFP, but substitutes glutamic acid for serine as the first amino acid residue in the tripeptide sequence.

Photoconversion of the Kaede/Eos Green-Red Highlighter Fluorophore - A variety of interesting and potentially very useful optical highlighters have been developed in fluorescent proteins cloned from reef coral and sea anemone species. One of the first and most important examples, a tetrameric fluorescent protein isolated from the stony Open Brain coral, Trachyphyllia geoffroyi, has been found to undergo photoconversion from green to red fluorescence emission upon irradiation with violet or ultraviolet light. The unusual and dramatic color transition prompted investigators to name the protein Kaede, after the color transition exhibited by leaves of the Japanese maple tree, which turn from green to red in the fall months. Subsequent investigations have uncovered additional green-red optical highlighters, including Kikume Green-Red and EosFP, derived from the stony corals Favia favus and Lobophyllia hemprichii, respectively. This interactive tutorial explores the molecular re-arrangement that occurs during the maturation of the Kaede fluorescent protein fluorophore, which emits green fluorescence, as well as the mechanism of photoconversion that cleaves the peptide backbone to yield a red fluorescent optical highlighter.

Contributing Authors

David W. Piston - Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, 37232.

Jennifer Lippincott-Schwartz and George H. Patterson - Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, 20892.

Matthew J. Parry-Hill, Nathan S. Claxton, Scott G. Olenych, and Michael W. Davidson - National High Magnetic Field Laboratory, 1800 East Paul Dirac Dr., The Florida State University, Tallahassee, Florida, 32310.


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