Selective Labeling of Proteins by Using Protein Farnesyltransferase

The challenging task of identifying and studying protein function has been greatly aided by labeling proteins with reporter groups. Here, we present a strategy that utilizes an enzyme that labels a four‐residue sequence appended onto the C terminus of a protein, with an alkyne‐containing substrate....

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Bibliographic Details
Published in:Chembiochem : a European journal of chemical biology 2007-01, Vol.8 (1), p.98-105
Main Authors: Duckworth, Benjamin P., Zhang, Zhiyuan, Hosokawa, Ayako, Distefano, Mark D.
Format: Article
Language:English
Subjects:
Alkyl and Aryl Transferases - chemistry
Biochemistry - instrumentation
Biochemistry - methods
Chromatography, High Pressure Liquid
click chemistry
Fluorescence Resonance Energy Transfer - methods
Fluorescent Dyes - pharmacology
FRET
Green Fluorescent Proteins - chemistry
Green Fluorescent Proteins - metabolism
Kinetics
Models, Chemical
Models, Molecular
Molecular Conformation
Molecular Probes
protein farnesyltransferase
Protein Folding
protein immobilization
protein modifications
Protein Structure, Tertiary
Proteins - chemistry
Spectrometry, Mass, Electrospray Ionization
Substrate Specificity
Time Factors
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Summary:The challenging task of identifying and studying protein function has been greatly aided by labeling proteins with reporter groups. Here, we present a strategy that utilizes an enzyme that labels a four‐residue sequence appended onto the C terminus of a protein, with an alkyne‐containing substrate. By using a bio‐orthogonal cycloaddition reaction, a fluorophore that carried an azide moiety was then covalently coupled to the alkyne appended on the protein. FRET was used to calculate a Förster (R) distance of 40 Å between the eGFP chromophore and the newly appended Texas Red fluorophore. This experimental value is in good agreement with the predicted R value determined by using molecular modeling. The small recognition tag, the high specificity of the enzyme, and the orthogonal nature of the derivatization reaction will make this approach highly useful in protein chemistry. Many methods exist to label proteins, but few offer the selectivity that is often required. This paper presents a new in vitro protein‐labeling strategy that offers selectivity. An enzyme is used to label a target protein with an alkyne‐containing molecule, which is then treated with an azide‐containing fluorophore.
ISSN:1439-4227
1439-7633
DOI:10.1002/cbic.200600340