Synthesis and biological activity of photoactivatable N-ras peptides and proteins

J Am Chem Soc. 2003 Oct 22;125(42):12749-58. doi: 10.1021/ja036178d.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzophenones / chemical synthesis
  • Benzophenones / chemistry
  • Cysteine / analogs & derivatives
  • Cysteine / chemistry
  • Oligopeptides / chemical synthesis
  • Oligopeptides / chemistry
  • Oligopeptides / pharmacology
  • PC12 Cells
  • Peptide Fragments / chemical synthesis*
  • Peptide Fragments / chemistry
  • Peptide Fragments / pharmacology
  • Photochemistry
  • Rats
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • ras Proteins / chemical synthesis*
  • ras Proteins / chemistry
  • ras Proteins / pharmacology

Substances

  • Benzophenones
  • Oligopeptides
  • Peptide Fragments
  • ras Proteins
  • Cysteine