A new intrinsic fluorescent probe for proteins Biosynthetic incorporation of 5-hydroxytryptophan into oncomodulin

• Published in: FEBS letters Vol. 310; no. 3; pp. 269 - 272
• Main Authors: Hogue, Christopher W.V., Rasquinha, Ingrid, Szabo, Arthur G., MacManus, John P.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 05-10-1992; Elsevier
• Subjects: 5-Hydroxytryptophan - metabolism; Alloprotein; Anisotropy; Biological and medical sciences; Calcium-Binding Proteins - immunology; Calcium-Binding Proteins - metabolism; Diverse techniques; Epitopes; Fluorescence anisotropy; Fluorescent Dyes - analysis; Fluorescent Dyes - metabolism; Fundamental and applied biological sciences. Psychology; Macromolecular Substances; Molecular and cellular biology; Protein fluorescence; Protein—protein interaction; Recombinant Proteins - biosynthesis; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Protein—protein interaction; Protein fluorescence; Fluorescence anisotropy; Alloprotein; Binding protein; Fluorescent tracer; Antibody; Escherichia coli; Bacteria; Molecular interaction; Molecular probe; Calcium Ions; Enterobacteriaceae
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/0014-5793(92)81346-N

The tryptophan analog, 5-hydroxytryptophan (5HW), has a significant absorbance between 310–320 nm, which allows it to act as an exclusive fluorescence probe in protein mixtures containing a large number of tryptophan residues. Here for the first time a method is reported for the biosynthetic incorporation of 5HW into an expressed protein, the Y57W mutant of the Ca
2+ binding protein, oncomodulin. Fluorescence anisotropy and time-resolved fluorescence decay measurements of the interaction between anti-oncomodulin antibodies and the 5HW-incorporated oncomodulin conveniently provide evidence of complex formation and epitope identification that could not be obtained with the natural amino acid. This report demonstrates the significant potential for the use or 5HW as an intrinsic probe in the study of structure and dynamics of protein—protein interactions.