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Labeling proteins with small molecules by site-specific posttranslational modification.

Jun Yin1, Fei Liu, Xiaohua Li

  • 1Department of Biological Chemistry and Molecular Pharmacology and Institute of Chemistry and Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA.

Journal of the American Chemical Society
|June 24, 2004
PubMed
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We developed a method for site-specific protein labeling using a peptide carrier protein (PCP) tag and the enzyme phosphopantetheinyl transferase Sfp. This strategy enables efficient and specific attachment of small molecules to target proteins within cell lysates.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Chemical Biology

Background:

  • Site-specific protein labeling is crucial for studying protein function and engineering novel biomolecules.
  • Existing methods often face limitations in specificity, efficiency, or compatibility with diverse protein targets and labeling molecules.

Purpose of the Study:

  • To develop a general and efficient strategy for site-specific protein labeling with small molecules.
  • To leverage the phosphopantetheinyl transferase Sfp and a peptide carrier protein (PCP) tag for targeted covalent modification.

Main Methods:

  • Proteins of interest were expressed as fusions to the peptide carrier protein (PCP) tag.
  • The enzyme phosphopantetheinyl transferase (Sfp) was used to catalyze the covalent attachment of small molecule-phosphopantetheinyl conjugates to a specific serine residue on the PCP tag.

Related Experiment Videos

  • Labeling reactions were performed on PCP fusion proteins in cell lysates.

    • Main Results:

    • The developed strategy achieved site-specific labeling of PCP fusion proteins with high specificity and efficiency.
    • The PCP tag demonstrated compatibility with a variety of proteins.
    • Sfp-catalyzed modification of the PCP tag was compatible with diverse small-molecule probes conjugated to coenzyme A.

    Conclusions:

    • The PCP tag and Sfp-catalyzed modification provide a versatile platform for site-specific protein labeling with small molecules.
    • This method holds significant potential for applications in chemical biology, drug discovery, and protein engineering.