A new intrinsic fluorescent probe for proteins. Biosynthetic incorporation of 5-hydroxytryptophan into oncomodulin.
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 of 5HW as an intrinsic probe in the study of structure and dynamics of protein-protein interactions.
Hogue
C W
CW
Division of Biological Science, National Research Council of Canada, Ottawa, Ont.
Rasquinha
I
I
Szabo
A G
AG
MacManus
J P
JP
D006916:5-Hydroxytryptophan / Q000378:metabolism*; D016880:Anisotropy; D002135:Calcium-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D000939:Epitopes; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism*; D046911:Macromolecular Substances; D011994:Recombinant Proteins / Q000096:biosynthesis; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D002135:Calcium-Binding Proteins; D000939:Epitopes; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C034733:oncomodulin; D006916:5-Hydroxytryptophan