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 transduction and vesicular transport. Although post-translational lipid modification of numerous proteins in signal transduction is crucial for biological function, analysis of protein-protein interactions has mainly focused on recombinant proteins in solution under defined in vitro conditions. Here we present a new strategy for the synthesis of such lipid-modified proteins. It involves the bacterial expression of a carboxy-terminally truncated non-lipidated protein, the chemical synthesis of differently lipidated peptides representing the C terminus of the proteins, and their covalent coupling. Our technique is demonstrated using Ras constructs, which exhibit properties very similar to fully processed Ras, but can be produced in high yields and are open for selective modifications. These constructs are operative in biophysical and cellular assay systems, showing specific recognition of effectors by Ras lipoproteins inserted into the membrane surface of biosensors and transforming activity of oncogenic variants after microinjection into cultured cells.
Bader
B
B
Max-Planck Institut für Molekulare Physiologie, Dortmund, Germany.
Kuhn
K
K
Owen
D J
DJ
Waldmann
H
H
Wittinghofer
A
A
Kuhlmann
J
J
D000818:Animals; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D050356:Lipid Metabolism; D008074:Lipoproteins / Q000096:biosynthesis / Q000378:metabolism; D008567:Membranes, Artificial; D016716:PC12 Cells; D011499:Protein Processing, Post-Translational; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015398:Signal Transduction; D018631:ras Proteins / Q000096:biosynthesis* / Q000378:metabolism
D008074:Lipoproteins; D008567:Membranes, Artificial; D011994:Recombinant Proteins; D018631:ras Proteins