Structural basis for dual excitation and photoisomerization of the aequorea victoria green fluorescent protein. Crystal structure of the aequorea victoria green fluorescent protein. Many cnidarians utilize greenfluorescent proteins gfps as energytransfer acceptors in bioluminescence. Its amazing ability to generate a highly visible, efficiently emitting internal. Properties of the gfp protein and its fluorescence chromophore the native green fluorescent protein gfp, first so named by morin and hastings 1971 ab.
The nucleotide sequences of the cdna and the gene will aid in the elucidation of structurefunction. Dissection of the structure of the protein would be expected to shed light on its potential applications to other fields such as the detection of protease activity. Green fluorescent protein gfp is a protein in the jellyfish aequorea victoria 8 that exhibits green fluorescence when exposed to light. Cormier 1992 primary structure of the aequorea victoria green. In living cells, this has been achieved by following fluorescence recovery after local microbeam photobleaching of microinjected fluorescently labelled protein see, for example, 1 as well as by photoactivation of caged fluorescently. Deletions of the aequorea victoria green fluorescent protein define. Green fluorescent protein and homologs the family of gfplike.
The green fluorescent protein gfp of the jellyfish aequorea victoria is an unusual protein with strong visible absorbance and fluorescence from a phydroxybenzylideneimidazolidinone chromophore, which is generated by cyclization and oxidation of the proteins own sertyrgly sequence at positions 6567. Green fluorescent protein gfp from aequorea victoria. Gfp green fluorescent protein aequorea victoria jellyfish gfp. Green fluorescent protein gfp is a protein in the jellyfish aequorea victoria that exhibits green fluorescence when exposed to light. Green fluorescent protein has been engineered to produce a vast number of variously colored mutants, fusion proteins, and biosensors. Gfps fluoresce in vivo upon receiving energy from either a luciferaseoxyluciferin excitedstate. The green fluorescent protein gfp from the jellyfish aequorea victoria is a widely used. Considerable insight into many cell biological processes can be obtained by following the turnover of individual protein species in time and space. The protein has 238 amino acids, three of them numbers 65 to 67 form a structure that emits visible green fluorescent light.
Gfp identified as protein, extracted from 10,000 jellyfish a protein giving solutions that look slightly greenish in sunlight though only yellowish under tungsten lights, and exhibiting a very bright, greenish fluorescence in the ultraviolet of a mineralite, has also been isolated from squeezates. Artistic rendition of green fluorescent protein gfp, based on pdb entry 1ema. Zeiss microscopy online campus aequorea fluorescent. He is known for his work to clone and sequence the genes for the photoprotein aequorin and green fluorescent protein gfp and for his proposal to use gfp as a tracer molecule. Theprotein fold consists of an 11stranded barrel with a. The molecular structure of green fluorescent protein pdf. Green fluorescent protein was discovered by shimomura et al 1 as a companion protein to aequorin, the famous chemiluminescent protein from aequorea jelly. Chemical nature of the light emitter of the aequorea green. Prasher born august 1951 is an american molecular biologist. Structure of the aequorea victoria green fluorescent protein. Regions of secondary structure beta strands and alpha helices are marked. Build a paper model of green fluorescent protein gfp.
It is reported to be a very rapidlymaturing weak dimer with high acid sensitivity. These highly fluorescent proteins are unique due to the chemical nature of their chromophore, which is comprised of modified amino acid aa residues within the polypeptide. The barrellike structure of gfp was also determined in the 1990s. We have developed a way of imaging metastases in mice by use of tumour cells expressing green fluorescent protein gfp that can be used to examine fresh tissue, both in situ and externally. Dissection of the structure of the protein would be expected to shed light on its potential. Excitation at the primary absorption peak of 395 nmyields an emission maximum at 508 nmwith a quantum.
Mapping gfp structure evolution during proton transfer. Green fluorescent protein imaging of tumour growth. Gfp, the green fluorescent protein from the jellyfish aequorea victoria, is widely used in the life sciences as a gene expression marker because of its efficient bioluminescence. Primary structure of the aequorea victoria greenfluorescent protein. The green fluorescent protein gfp is a protein composed of 238 amino acid residues 26. The chromophore, resulting from the spontaneous cyclization a. It is reported to be a very rapidlymaturing monomer with moderate acid sensitivity. Using optimized combinatorial mutagenisis techniques and digital imaging spectroscopy dis, we have insulated mutants of the cloned aequorea victoria. Green fluorescent protein can be mutated to emit at different wavelengths such as blue for bfp when tyr66 is replaced by his, cyan for cfp when tyr66 is replaced by trp, and yellow for yfp when thr203 is replaced by tyr. Green fluorescent protein wikipedia republished wiki 2. The wild type wt aequorea gfp displays a complex absorption spectrum, with maximal excitation occurring at 397 nanometers, and a minor secondary peak of residing at 476 nanometers. Although many other marine organisms have similar green fluorescent proteins, gfp traditionally refers to the protein first isolated from the jellyfish aequorea victoria.
Eyfp is a basic constitutively fluorescent greenyellow fluorescent protein published in 1996, derived from aequorea victoria. There is inconsistency in the literature regarding the. The green fluorescent protein is a bioluminescent protein that is commonly found in the aequorea victoria jellyfish and most recently found in the first ever reptile, the hawsbill sea turtle. The purpose of both the primary bioluminescence from aequorins action on luciferin and the. Primary structure of the aequorea victoria greenfluorescent protein author douglas c. Ground and photoactive states of the protonwire in the green fluorescent protein.
Redshifted excitation mutants of the green fluorescent. Green fluorescent protein samantha wilkison, corey lane. Green fluorescent protein gfp is the founding member of a new class of proteins, the. We have now also introduced the gfp gene, cloned from. The green fluorescent protein gfp from the jellyfish aequorea victoria is the first known protein in which visible fluorescence is genetically encodable. The chromophore, resulting from the spontaneous cyclization and oxidation of the sequence ser65 or thr65tyr66gly67, requires the native protein fold for both formation and fluorescence emission. Crystal structure of the aequorea victoria green fluorescent protein mats ormo, andrew b. This report describes the cloning and sequencing of both cdna and genomic clones of gfp from the cnidarian, aequorea victoria.
Wavelength mutations and posttranslational autoxidation of. The protein is made up of 238 amino acid residues in a single polypeptide chain and produces a greenish fluorescence. Primary amino acid sequence of gfp indicates a glu residue at this position. Green fluorescent protein gfp was first isolated from the jellyfish aequorea victoria in the early 1960s. The aequorea victoria green fluorescent protein gfp is widely recognized as a powerful tool in cell biology, serving as a vital reporter for monitoring localization and dynamics of intracellular. Operation of the proton wire in green fluorescent protein. In the 1990s, methods were developed to express the gfp gene in worms and other organisms. A number of investigators have examined the dynamics of wtgfp protein in the excited state, and showed that fluorescence resulted from. The green fluorescent protein gfp from the pacific northwest jellyfish aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. He communicated his pioneering work to martin chalfie and roger y.
The fluorophore is derived from natural residues present within the primary structure of gfp, so no exogenous cofactor or substrate is needed for fluorescence 1, 2. These mice present many new possibilities for research including realtime studies of tumour progression, metastasis, and drugresponse evaluations. We use cookies to offer you a better experience, personalize content, tailor advertising, provide social media features, and better understand the use of our services. The molecular structure of green fluorescent protein. Chemical structure of the hexapeptide chromophore of the. Original report on the characterization of gfp from aequorea victoria referred to as gfp10, which encodes a 238amino acid polypeptide with a calculated molecular weight of 26,888. Pdf primary structure of the aequorea victoria green. Upon mechanical stimulation of the organism, gfp emits green light spectrally identical to its fluorescence. Journal of chemical theory and computation 2008, 4 7, 181150. Volume 111, issue 2, 15 february 1992, pages 229233. Pdf primary structure of the aequorea victoria greenfluorescent.
Green fluorescent substance in jellyfish first described. The greenfluorescent proteins gfp are a unique class of proteins involved in bioluminescence of many cnidaria. Primary structure of the aequorea victoria greenfluorescent protein author links open overlay panel douglas c. Examination of listeria monocytogenes intracellular gene expression by using the green fluorescent protein of aequorea victoria. James remingtont the green fluorescent protein gfp from the pacific northwest jellyfish aequorea victoria has generated intense interest as a marker for gene expression and localization of gene products. The green fluorescent protein gfp from the jellyfish aequorea victoria is a widely used marker for gene expression and protein localization studies. Tsien, but by 1991 was himself unable to obtain further research funding, and. Deletions of the aequorea victoria green fluorescent. In just three years, the green fluorescent protein gfp from the jellyfish aequorea victoria has vaulted from obscurity to become one of the most widely studied and exploited proteins in biochemistry and cell biology. We constructed gfp h, a synthetic version of the jellyfish aequorea victoria green fluorescent protein gfp cdna that is adapted for highlevel expression in mammalian cells, especially those of human origin.
1512 1124 1317 1223 1359 68 1268 484 363 1461 311 1128 1405 828 1137 1109 738 664 461 525 793 150 454 500 345 1379 1268 720 683 1531 801 877 253 1371 1122 1317 1182 482 269 532 1243 647 1469 409