Fluorescence lifetimes of the tryptophan residues in ornithine transcarbamoylase.
Multifrequency (2-230 MHz) phase-modulation fluorescence measurements and site-directed mutagenesis have been employed to assign fluorescence lifetimes, quantum yields, and emission maxima to the four tryptophans in the enzyme ornithine transcarbamoylase from Escherichia coli (OTCase) (Trp-125, -92, -233, and -243). OTCase displays two apparent fluorescence lifetimes, 7.2 and 3.2 ns. Results on specific mutants show that Trp-233 has a lifetime of 7.1 ns, while TRP-125, -192, and -243 have lifetimes of 4.0, 3.6, and 4.9 ns, respectively. Thus, the specific conformational changes of the polypeptide segment involving Trp-233 may be monitored conveniently in the wild-type enzyme. On the basis of quantum yield values, Trp-233 is calculated to contribute approximately 43% of the fluorescence intensity of the enzyme, while direct measurements of the enzyme show that up to 65% of the total intensity is really emitted by this tryptophan. The discrepancy may arise from energy transfer from Trp-125 to Trp-233, with an efficiency of 20%. Application of the assigned tryptophan lifetimes to probe ligand-induced protein conformational changes has also been demonstrated.
Shen
W H
WH
Department of Chemistry, Boston University, Massachusetts 02215.
D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009954:Ornithine Carbamoyltransferase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D014364:Tryptophan; D009954:Ornithine Carbamoyltransferase