A Photoactivatable Prenylated Cysteine Designed to Study Isoprenoid Recognition

Abstract

Protein prenylation, involving the alkylation of a specific C-terminal cysteine with a C(15) or C(20) isoprenoid unit, is an essential posttranslational modification required by most GTP-binding proteins for normal biological activity. Despite the ubiquitous nature of this modification and numerous efforts aimed at inhibiting prenylating enzymes for therapeutic purposes, the function of prenylation remains unclear. To explore the role the isoprenoid plays in mediating protein-protein recognition, we have synthesized a photoactivatable, isoprenoid-containing cysteine analogue (2) designed to act as a mimic of the C-terminus of prenylated proteins. Photolysis experiments with 2 and RhoGDI (GDI), a protein which interacts with prenylated Rho proteins, suggest that the GDI is in direct contact with the isoprenoid moiety. These results, obtained using purified GDI as well as Escherichia coli (E. coli) crude extract containing GDI, suggest that this analogue will be an effective and versatile tool for the investigation of putative isoprenoid binding sites in a variety of systems. Incorporation of this analogue into peptides or proteins should allow for even more specific interactions between the photoactivatable isoprenoid and any number of isoprenoid binding proteins.

Synthesis of high specific activity 35S‐labelled N‐methanesulfonyl farnesylcysteine and a photoactive analog

“…21 Examination of Figure 2, showing the superpositioning of [ 35 S]4 with the crystal structure of only the geranylgeranylated cysteine residue of Cdc42, reveals good complementarity between the structure of 4 and the conformation of the bound geranylgeranyl group. The preparation of bromide 8 (Scheme 2) was reported in earlier work, 41,57 and the alkylation of cysteine methyl ester under basic conditions to yield 9 was accomplished as described for the farnesylated analogs above. 41 The following reactions, using first [ 35 Furthermore, in comparison with a previously described preparation of these materials, 41 an improved purity of [ 35 S]3 was obtained and the radiochemical yield of [ 35 S]4 was greatly increased while a high degree of radiochemical purity was preserved.…”

“…The preparation of bromide 8 (Scheme 2) was reported in earlier work, 41,57 and the alkylation of cysteine methyl ester under basic conditions to yield 9 was accomplished as described for the farnesylated analogs above. 41 The following reactions, using first [ 35 Furthermore, in comparison with a previously described preparation of these materials, 41 an improved purity of [ 35 S]3 was obtained and the radiochemical yield of [ 35 S]4 was greatly increased while a high degree of radiochemical purity was preserved. Figure 3 shows the HPLC chromatograms of the radiolabelled benzophenone-containing prenylcysteines, the retention times of which matched those of the unlabelled counterparts.…”

“…41 Because of the preponderance of C-terminal cysteine carboxymethylated prenylated proteins, 4,11,13 the ester precursor to this prenylcysteine free acid, [ 35 S]3, was examined for its cross-linking abilities to GDI in several contexts. Purified GDI 62,63 was first irradiated in the presence of the esterified probe [ 35 S]3, followed by competition experiments with AGGCE and AGGC.…”

“…The rationale for this modification was based on work reported by Dean et al 42 who showed that methanesulfonyl chloride incorporating an 35 S radiolabel could be used to generate high specific activity (1100 Ci/mmol) compounds from amine precursors. In our initial work in this area, 41 we prepared a photoactive analog, [ 35 S]4, of N-acetyl farnesylcysteine, 2b, that contained a benzophenone photophore and an 35 S radiolabel. Compound [ 35 S]4 was used in crosslinking experiments and was shown to label the protein RhoGDI (Rho GDP-dissociation inhibitor) at physiological meaningful concentrations (51 mM).…”

Use of fluorous and solid-phase electrophiles as scavengers for excess amine in the preparation of sulfur-35 labelled radioligands

“…[3][4][5] Typically, 10-50 mCi of the appropriate carrier-free [ 35 S]sulfonyl chloride (1-10 mg, $1000 Ci/mmol) is reacted with a large excess of precursor amine (2-5 mg). Purification is then conducted using semipreparative HPLC.…”