Synthesis and biological activity of photoactivatable N-ras peptides and proteins.
A modular strategy for the assembly of farnesylated N-Ras heptapeptides carrying a photoactivatable benzophenone (BP) group within the lipid residue is described. This strategy is based on the fragment condensation of a N-terminal hexapeptide synthesized on the solid support with a cysteine methyl ester which is modified with different farnesyl analogues, incorporating the photophor. At the N-terminus of the peptides different functional groups can be attached, e.g., biotin for product enrichment and detection after photoactivation or a maleimido (MIC) linker, allowing for the coupling to proteins carrying a C-terminal free cysteine. Using this strategy, 24 peptides were synthesized, incorporating farnesyl analogues with four different chain lengths. Two of these photoactivatable conjugates were ligated to oncogenic human N-RasG12V Delta 181. A cellular transformation assay revealed that the semisynthetic proteins retain their biological activity despite the photolabel. The first photolabeling experiments with a geranyl-BP-labeled N-Ras construct and the farnesyl-sensitive guanine nucleotide exchange factor hSos1 indicate that this photoaffinity labeling system can be particularly useful for studying protein-protein interactions, e.g., the participation of the farnesyl group in Ras signaling, which is still discussed with controversy.
Völkert
Martin
M
Abteilung Chemische Biologie, Max-Planck-Institut für Molekulare Physiologie, and Organische Chemie, Universität Dortmund, D-44227, Germany.
Uwai
Koji
K
Tebbe
Andreas
A
Popkirova
Boriana
B
Wagner
Melanie
M
Kuhlmann
Jürgen
J
Waldmann
Herbert
H
D000818:Animals; D001577:Benzophenones / Q000138:chemical synthesis / Q000737:chemistry; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D016716:PC12 Cells; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D010777:Photochemistry; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D018631:ras Proteins / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology
D001577:Benzophenones; D009842:Oligopeptides; D010446:Peptide Fragments; D018631:ras Proteins; D003545:Cysteine