Introduction by site-directed mutagenesis of a tryptophan residue as a fluorescent probe for the folding of Escherichia coli phosphofructokinase.
The leucine residue at position 178 in the major allosteric phosphofructokinase from Escherichia coli has been replaced by a tryptophan using site-directed mutagenesis. Transformation by the mutated gene of pfk- bacteria results into the expression of a pfk+ phenotype and the production of an active enzyme. The modified protein has been purified and its fluorescence properties show that it contains 2 tryptophan residues, the original Trp 311 and the new Trp 178. During unfolding of the protein by guanidine hydrochloride, the changes in the fluorescence of these 2 residues take place at different steps: Trp 311 becomes exposed to solvent when the dimeric form dissociates into monomers, while Trp 178 is exposed only when a folded chain loses its tertiary structure. The mutant enzyme is stabilized by its substrate fructose-6-phosphate against denaturation induced by heat or guanidine hydrochloride.
Teschner
W
W
Immuno AG, Vienna, Austria.
Serre
M C
MC
Garel
J R
JR
D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D010957:Plasmids; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000235:genetics
D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D014364:Tryptophan; D010732:Phosphofructokinase-1