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. 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 A 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.
Duckworth
Benjamin P
BP
Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Zhang
Zhiyuan
Z
Hosokawa
Ayako
A
Distefano
Mark D
MD
D019883:Alkyl and Aryl Transferases / Q000737:chemistry*; D001671:Biochemistry / Q000295:instrumentation / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D005456:Fluorescent Dyes / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008968:Molecular Conformation; D015335:Molecular Probes; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity; D013997:Time Factors
D005456:Fluorescent Dyes; D015335:Molecular Probes; D011506:Proteins; D049452:Green Fluorescent Proteins; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase