29] Structural analysis of protein prenyl groups and associated C-terminal modifications

Abstract

The prenylation of proteins, including several in the visual transduction pathway, is one of the most recently discovered modifications of eukaryotic cell proteins.1–4 Some prenylated proteins, including rhodopsin kinase, the γ subunit of transducin, the α subunit of retinal cGMP phosphodiesterase, Ras, and lamin B, are initially produced with a C-terminal sequence motif CaaX (C is cysteine, a is usually but not necessarily an aliphatic residue, and X is typically M, S, or Q). Prenylation occurs by enzyme-catalyzed attachment of a 15-carbon farnesyl group to cysteine via a thioether linkage. After prenylation, aaX is released by a membrane-bound endoprotease,5–8 and the newly exposed S-farnesylcysteine is methylated on its α-carboxyl group by a membrane-bound methyltransferase.9,10 Other prenylated proteins, including the γ subunits of many heterotrimeric G proteins, the β subunit of retinal cGMP phosphodiesterase, and a subset of small GTP-binding proteins, contain a CaaX motif (X = L, F) and are modified by the attachment of the 20-carbon geranylgeranyl group. Removal of aaX and C-terminal methylation occurs as for farnesylated proteins. Finally, the subset of GTP-binding proteins termed Rab contains two cysteines near or at the C terminus (CCXX, XXCC, CXC), and both cysteine sulfhydryls contain a thioether-linked geranylgeranyl group.11 The attachment of prenyl groups to these three classes of proteins is catalyzed by three distinct protein prenyltranferases.12,13 Structures of protein prenyl groups have been rigorously established by releasing the protein-bound lipids with Raney nickel (to cleave reductively the bond between cysteine S and C-1 of the prenyl group) and analyzing the released hydrocarbons by combined gas chromatography–mass spectrometry versus authentic standards.14–17 Protein prenyl groups can also be released by treatment with methyl iodide to produce farnesol and geranylgeraniol along with isomerized prenols.16,18 This method has been used when relatively small amounts of prenylated protein are available, typically from tissue culture cells that have been grown in the presence of radiolabeled mevalonic acid (the precursor of prenyl groups) or its lactone in the presence of statins, which block the production of endogenous mevalonic acid. The radiolabeled prenol is analyzed versus standards by reversed phase high-performance liquid chromatography (HPLC).16 While this approach does not establish the chemical structure of the prenyl group, it provides strong circumstantial evidence for the presence of protein-bound farnesyl and geranylgeranyl groups. However, in our hands, we have experienced difficulties with methyl iodide cleavage, in that yields are highly variable and can be quite low in some cases (<10% cleavage). Work in our laboratory has shown that the Raney nickel cleavage method is superior to methyl iodide treatment, and the details of this procedure, combined with HPLC analysis of the released radiolabeled hydrocarbons, are described in this chapter. This method has been used to analyze protein prenyl groups from protein extracted from sodium dodecyl sulfate (SDS)–polyacrylamide gel slices. Also described here is the use of Raney nickel cleavage followed by combined gas chromatography–mass spectrometry to determine the structure of prenyl groups released from delipidated total cell protein. The rigorous structural analysis of aaX removal and C-terminal methylation has been carried out by fast atom bombardment mass spectrometry of C-terminal peptides from prenylated proteins.11,19 A less rigorous approach has been used to explore C-terminal methylation: HPLC analysis of the product of oxidative scission of the prenyl–cysteine linkage combined with amide bond hydrolysis to yield cysteic acid methyl ester.20 In the present chapter, a method for electrospray mass spectrometric analysis of protein C-terminal peptides is presented, which has been applied to the analysis of prenylated peptides in a complex mixture (a tryptic digest of partially purified bovine rhodopsin kinase). This method is facilitated by the availability of prenylated peptide standards, and a convenient synthesis of peptides and peptide methyl esters radiolabeled with high specific activity farnesyl and geranylgeranyl groups is described.