Selective Labeling of Proteins by Using Protein Farnesyltransferase

Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
ChemBioChem (Impact Factor: 3.09). 01/2007; 8(1):98-105. DOI: 10.1002/cbic.200600340
Source: PubMed


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.

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    • "Figure 3b shows the FRET histogram when 2.7 kb TxY-DNA is packaged in the GFP-portal. From this measurement, the donor-to-acceptor distance (r) is estimated to be ∼ 5.8 nm, given a Förster distance of 4 nm for a GFP-Txred donor-acceptor pair (Duckworth et al., 2007). As previously concluded, evidently the 2.7 kb leader portion of the DNA substrate serves to introduce and anchor the DNA within the procapsid (Oram, Sabanayagam, and Black, 2008). "
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    • "Genetic encoding of proteins of interest with a FP is the most popular labeling method in cells (Tsien, 1998). Other modes of attachment of exogenous fluorophores to cellular proteins are based on (i) enzymatic strategies such as acyl carrier protein/phosphopantetheine transferase (George et al., 2004); Q-tag/transglutaminase (Lin and Ting, 2006), biotin acceptor peptide/biotin ligase (Howarth et al., 2005), prokaryotic hydrolase/Halo tag (Los et al., 2008), formylglycine-generating enzyme/aldehyde tag (Carrico et al., 2007), and Farnesyltransferase/CVIA peptide tag (Duckworth et al., 2007); and (ii) noncovalent affinity labeling methods such as the tetracysteine/biarsenical system (Andresen et al., 2004; Hoffmann et al., 2005), dihydrofolate reductase (DHFR) (Miller et al., 2004, 2005), the bis-arsenical/SplAsH (spirolactam Arsenical Hairpin binder) system (Bhunia and Miller, 2007), biotin/streptavidin (Weber et al., 1989), and barnase/barstar (Wang et al., 2004). QDs have emerged as a very promising class of fluorophores for multiple biological applications (Michalet et al., 2005), including live cell imaging. "
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