Chemoenzymatic Synthesis of Biotinylated Ras Peptides and Their Use in Membrane Binding Studies of Lipidated Model Proteins by Surface Plasmon Resonance

Abstract

Characteristic peptides, which have the same lipid modifications as their parent proteins and labels, by which they can be traced, for example, the biotin group, are efficient reagents for the study of signal transduction processes using lipid-modified Ras pro- teins. Such peptide conjugates often contain both acid- and base-labile groups, and their synthesis calls for the application of protecting groups that can be removed selectively and under ex- tremely mild conditions. These criteria are met by the enzyme-labile choline (Cho) ester group. Choline esters can be cleaved at pH 6.5 and room temperature by employing the enzyme butyrylcho- line esterase from horse serum. By using this enzymatic protecting group techni- que as the key step, access to the characteristic S-palmitoylated and S-far- nesylated C terminus of the human N- Ras protein was successfully achieved. The conditions under which these enzy- matic deprotections proceed are so mild that no undesired side reaction is ob- served (that is no hydrolysis or b elim- ination of the thioester and no acid- mediated attack on the double bonds of the farnesyl group). In addition to this technique, the allyl ester group was removed by means of Pd 0 -mediated allyl transfer to accepting nucleophiles. This reaction was exploited to construct bio- tin-labeled lipidated peptides, which correspond to the C terminus of N-Ras, and labeled analogues thereof. The lipi- dated and biotinylated peptides served as anchors for a protein moiety in an artifical membrane in a BIAcore surface plasmon resonance system. The stability of the membrane insertion was moni- tored by surface plasmon resonance after the binding of streptavidin to the biotin heads of farnesylated or farnesy- lated and palmitoylated peptides. Thus, it was shown that a double hydrophobic modification of the peptides is necessary to obtain stable insertion of the conju- gates.