A minimalist substrate for enzymatic peptide and protein conjugation

Chembiochem. 2009 Dec 14;10(18):2934-43. doi: 10.1002/cbic.200900566.

Abstract

Recently a number of nonnatural prenyl groups containing alkynes and azides have been developed as handles to perform click chemistry on proteins and peptides ending in the sequence "CAAX", where C is a cysteine that becomes alkylated, A is an aliphatic amino acid and X is any amino acid. When such molecules are modified, a tag containing a prenyl analogue and the "CAAX box" sequence remains. Here we report the synthesis of an alkyne-containing substrate comprised of only nine nonhydrogen atoms. This substrate was synthesized in six steps from 3-methylbut-2-en-1-ol and has been enzymatically incorporated into both proteins and peptides by using protein farnesyltransferase. After prenylation the final three amino acids required for enzymatic recognition can be removed by using carboxypeptidase Y, leaving a single residue (the cysteine from the "CAAX box") and the prenyl analogue as the only modifications. We also demonstrate that this small tag minimizes the impact of the modification on the solubility of the targeted protein. Hence, this new approach should be useful for applications in which the presence of a large tag hinders the modified protein's solubility, reactivity, or utility.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alkyl and Aryl Transferases / chemistry*
  • Alkyl and Aryl Transferases / metabolism
  • Alkynes / chemistry
  • Azides / chemistry
  • Cathepsin A / metabolism
  • Diphosphates / chemical synthesis
  • Diphosphates / chemistry*
  • Kinetics
  • Peptides / chemistry*
  • Peptides / metabolism
  • Protein Prenylation
  • Substrate Specificity

Substances

  • Alkynes
  • Azides
  • Diphosphates
  • Peptides
  • Alkyl and Aryl Transferases
  • geranylgeranyltransferase type-I
  • p21(ras) farnesyl-protein transferase
  • Cathepsin A