PMID:8268168
| Citation |
Shen, WH (1993) Fluorescence lifetimes of the tryptophan residues in ornithine transcarbamoylase. Biochemistry 32:13925-32 |
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| Abstract |
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. |
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| Keywords |
Amino Acid Sequence; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Ornithine Carbamoyltransferase/chemistry; Ornithine Carbamoyltransferase/genetics; Ornithine Carbamoyltransferase/isolation & purification; Protein Conformation; Spectrometry, Fluorescence; Tryptophan/chemistry |
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