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

FEBS Letters

MacManus, John P.¹  Rasquinha, Ingrid¹  Szabo, Arthur G.¹  Hogue, Christopher W.V.¹ 

[1] Division of Biological Science, National Research Council of Canada, Ottawa, Ont. K1A 0R6, Canada

关键词: Protein fluorescence;    Fluorescence anisotropy;    Protein—protein interaction;    Alloprotein;   

DOI  :  10.1016/0014-5793(92)81346-N

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 Ca²⁺ 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.

【 授权许可】

Unknown