UNIT 26.1 Misincorporation Proton-Alkyl Exchange (MPAX): Engineering Cysteine Probes into Proteins

  1. Alondra Schweizer Burguete,
  2. Pehr B. Harbury,
  3. Suzanne R. Pfeffer

Published Online: 1 DEC 2005

DOI: 10.1002/0471140864.ps2601s42

Current Protocols in Protein Science

Current Protocols in Protein Science

How to Cite

Burguete, A. S., Harbury, P. B. and Pfeffer, S. R. 2005. Misincorporation Proton-Alkyl Exchange (MPAX): Engineering Cysteine Probes into Proteins. Current Protocols in Protein Science. 42:26.1:26.1.1–26.1.23.

Author Information

  1. Stanford School of Medicine, Stanford, California

Publication History

  1. Published Online: 1 DEC 2005
  2. Published Print: NOV 2005


This unit describes a rapid and efficient method to screen a polypeptide for amino acid residues that contribute to protein-protein interaction interfaces. Cysteine residues are introduced as positional probes in a protein at random by co-expression in bacteria with specific cysteine misincorporator tRNAs. The protein is then purified as an ensemble of polypeptides containing cysteine at low frequency, at different positions in each molecule. The ability of the native protein structure to protect different cysteine residues from chemical modification by iodoacetamide is determined to obtain a protein surface map that reveals candidate surface residues that are likely to be important for protein-protein interaction. Cysteine mutants with altered ligand binding can also be selected simultaneously by affinity chromatography.


  • Protein-protein interaction;
  • solvent accessibility;
  • protein structure;
  • cysteine misincorporation;
  • cysteine mutagenesis;
  • cysteine alkylation