Bioorganic synthesis of lipid-modified proteins for the study of signal transduction

Biological membranes define the boundaries of the cellular compartments in higher eukaryotes and are active in many processes such as signal transduct ion and vesicular transport. Although post-translational lipid modification of numerous proteins in signal transduction is crucial for biological func tion(1), analysis of protein-protein interactions has mainly focused on rec ombinant proteins in solution under defined in vitro conditions. Here we pr esent a new strategy for the synthesis of such lipid-modified proteins. It involves the bacterial expression of a carboxy-terminally truncated non-lip idated protein, the chemical synthesis of differently lipidated peptides re presenting the C terminus of the proteins, and their covalent coupling. Our technique is demonstrated using Ras constructs, which exhibit properties v ery similar to fully processed Ras, but can be produced in high yields and are open for selective modifications. These constructs are operative in bio physical and cellular assay systems, showing specific recognition of effect ers by Ras lipoproteins inserted into the membrane surface of biosensors an d transforming activity of oncogenic variants aft er microinjection into cu ltured cells.