Sequencing, proteins cysteine modification

In normal cells, the GDP/GTP-binding proteins, after protein synthesis, move to the cell membrane to which they become hooked by a hydrophobic farnesyl group. The y-subunit is anchored in the membrane by a post-translational modification of the C-terminal CAAX sequence (C - cystein, AA - aliphatic amino acids, X - methionine). This protein is first enzymatically farnesylated by a specific farnesyltransferase, then the AAX part is cleaved by specific proteases and finally the cystein residue is converted to a methyl ester. 

Acylation and prenylation. The amino terminus (usually glycine) of the a subunit of any G protein is nearly always converted to an N-myristoyl group.220-223 This modification occurs in a cotranslational process after removal of the initiating methionine (Chapter 29) and can be described as an acyl transfer from coenzyme A.224 The C termini of the y subunits of heterotrimeric G proteins, and also of the monomeric proteins of the Ras family, also undergo processing. For example, the C-terminal end of an intact Ras protein contains 18 residues which probably assume a largely a-helical conformation. A cysteine side chain near the terminus and having the sequence CAAX is... 

Prenylated proteins have characteristic C-terminal sequences. For example, the three allelic Ras proteins (H-Ras, K-Ras, and N-Ras) expressed in mammalian tissues contain a C-terminal tetrapeptide which begins with cysteine, and ends with either methionine or serine. This part of the molecule is referred to as the CaaX box where C = cysteine, a = an aliphatic amino acid, and X = a prenylation specificity residue. The first step in the posttranslational processing of Ras proteins utilizes FTase and farnesyl diphosphate (FPP) to covalently attach a farnesyl group to the cysteine thiol of the CaaX box. While subsequent processing events involve proteolytic removal of the aaX tripeptide and methylation of the resulting C-termi-nal carboxylate group, only the farnesyl modification is required for mutant Ras proteins to associate with the cell membrane and transform a cell.2-6... 

We chose H-RAS as the first target protein for several reasons. RAS proteins are, perhaps, the most intensively studied and best understood signal transduction proteins from the perspective of structure and function [8], The H-RAS crystal structure has been determined to the level of 1.5 A, and the domains involved in guanine nucleotide binding and hydrolysis, effector interactions, interactions with regulating proteins, and processing have been extensively characterized. RAS proteins are initially synthesized in the cytoplasm where they undergo a series of post-translational modifications at their carboxy-terminal sequence, the CaaX-box (where C is cysteine, a an aliphatic amino acid, and X either serine or... 

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