Deletions of the Aequorea victoria green fluorescent protein define the minimal domain required for fluorescence.
The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria is a widely used marker for gene expression and protein localization studies. 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. Using deletion analysis, we have defined the minimal domain in GFP required for fluorescence to amino acids 7-229. This domain starts at the middle of the first small alpha helix at the N terminus of GFP and ends immediately following the last beta sheet. Studies of the amino acids at both termini of the minimal domain revealed that positions 6 and 7 at the N terminus are Glu-specific. Change of the Glu residues to other amino acids results in reduction of GFP fluorescence. Position 229 at the C terminus of GFP, however, is nonspecific: the Ile can be replaced with other amino acids with no measurable loss of fluorescence. A total of only 15 terminal amino acids can be deleted from GFP without disrupting fluorescence, consistent with findings of a previous study of GFP crystal structure (Ormo, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y., Remington, S. J. (1996) Science 273, 1392-1395 and Yang, F., Moss, L. G., and Phillips, G. N., Jr. (1996) Nat. Biotechnol. 14, 1246-1251) that a tightly packed structure exists in the protein. We also generated internal deletions within the loop regions of GFP according to its crystal structure and found that all such deletions eliminated GFP fluorescence.
Li
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CLONTECH Laboratories, Inc., 1020 East Meadow Circle, Palo Alto, CA 94303, USA. xqli@CLONTECH.com
Zhang
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Ngo
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Zhao
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Kain
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Huang
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D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D005434:Flow Cytometry; D005453:Fluorescence; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D037401:Scyphozoa; D017384:Sequence Deletion; D014162:Transfection
D008164:Luminescent Proteins; D007532:Isoleucine; D049452:Green Fluorescent Proteins; D018698:Glutamic Acid