TY - JOUR
T1 - Selective labeling of proteins by using protein farnesyltransferase
AU - Duckworth, Benjamin P.
AU - Zhang, Zhiyuan
AU - Hosokawa, Ayako
AU - Distefano, Mark D.
PY - 2007/1
Y1 - 2007/1
N2 - 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.
AB - 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.
KW - Click chemistry
KW - FRET
KW - Protein farnesyltransferase
KW - Protein immobilization
KW - Protein modifications
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U2 - 10.1002/cbic.200600340
DO - 10.1002/cbic.200600340
M3 - Article
C2 - 17133644
AN - SCOPUS:33846333918
SN - 1439-4227
VL - 8
SP - 98
EP - 105
JO - ChemBioChem
JF - ChemBioChem
IS - 1
ER -