Immobilized and site-specifically labeled proteins are becoming invaluable tools in proteomics. Here, we describe a strategy to attach a desired protein to a solid surface in a covalent, site-specific manner. This approach employs an enzymatic posttranslational modification method to site-specifically label a target protein with an azide; an alternative substrate for protein farnesyl transferase containing an azide group was developed for this purpose. A bio-orthogonal Cu(I)-catalyzed cycloaddition reaction is then used to covalently attach the protein to agarose beads bearing an alkyne functional group. We demonstrate that both the azide incorporation and the capture steps can be performed on either a purified protein target or on a protein present within a complex mixture. This approach involves the use of a four-residue tag which is significantly smaller than most other tags reported to date and results in covalent immobilization of the target protein. Hence it should have significant applicability in protein science.

Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranylazide (C10) or farnesylazide (C15) moiety from the corresponding prenyldiphosphates to a model peptide substrate, N-dansyl-Gly-Cys-Val-Ile-Ala-OH. The rates of incorporation for these two substrate analogs are comparable and approximately twofold lower than that using the natural substrate farnesyl diphosphate (FPP). Reaction of N-dansyl-Gly-Cys(S-farnesylazide)-Val-Ile-Ala-OH with 2-diphenylphosphanylbenzoic acid methyl ester then gives a stable alkoxy-imidate linked product. This result suggests future generations whereby azide groups introduced using this enzymatic approach are functionalized using a broad range of azide-reactive reagents. Thus, chemistry has been developed that could be used to achieve highly specific peptide and protein modification. The farnesylazide analog may be useful in certain biological studies, whereas the geranylazide group may be more useful for general protein modification and immobilization.

Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranyl azide moiety to a peptide substrate, N-dansyl-Gly-Cys-Val-Ile-Ala-OH. The resulting azide-containing peptide was derivatized with a triphenylphosphine-based reagent to generate an O-alkyl imidate-linked product, rather than the amide-linked material expected via a Staudinger reaction. Since the CAAX box recognition motif (where the internal A residues are aliphatic amino acids) modified by PFTase can be incorporated into the C-terminus of virtually any polypeptide, this two-step procedure provides a general method for incorporating a diverse range of chemical modifications specifically near the C-terminus of proteins.

Originally designed to block the prenylation of oncogenic Ras, inhibitors of protein farnesyltransferase currently in pre-clinical and clinical trials are showing efficacy in cancers with normal Ras. Blocking protein prenylation has also shown promise in the treatment of malaria, Chagas disease, and progeria syndrome. A better understanding of the mechanism, targets and in vivo consequences of protein prenylation are needed to elucidate the mode of action of current PFTase inhibitors and to create more potent and selective compounds. Caged enzyme substrates are useful tools for understanding enzyme mechanism and biological function. Reported here is the synthesis and characterization of caged substrates of PFTase. The caged isoprenoid diphosphates are poor substrates prior to photolysis. The caged CAAX peptide is a true catalytically caged substrate of PFTase in that it is to not a substrate, yet is able to bind to the enzyme as established by inhibition studies and x-ray crystallography. Irradiation of the caged molecules with 350 nm light readily releases their cognate substrate, and their photolysis products are benign. These properties highlight the utility of those analogues towards a variety of in vitro and in vivo applications.