DELETIONS OF THE AEQUOREA-VICTORIA GREEN FLUORESCENT PROTEIN DEFINE THE MINIMAL DOMAIN REQUIRED FOR FLUORESCENCE

The Green Fluorescent Protein (GFP) from the jellyfish Aequorea victor ia is a widely used marker for gene expression and protein localizatio n studies. Dissection of the structure of the protein would be expecte d to shed light on its potential applications to other fields such as the detection of protease activity. Using deletion analysis, we have d efined the minimal domain in GFP required for fluorescence to amino ac ids 7-229. This domain starts at the middle of the first small cy heli x at the N terminus of GFP and ends immediately following the last bet a sheet. Studies of the amino acids at both termini of the minimal dom ain 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 De can be replaced with other amino acids with no measurable loss of fluorescence. A total of only 15 terminal amino aci ds 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 Philli ps, G. N., Jr. (1996) Not. Biotechnol. 14, 1246-1251) that a tightly p acked structure exists in the protein. We also generated internal dele tions within the loop regions of GFP according to its crystal structur e and found that all such deletions eliminated GFP fluorescence.