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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Updated: Jun 21, 2025

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking
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High-Performance Workflow for Identifying Site-Specific Crosslinks Originating from a Genetically Incorporated,

Lindsey D Ulmer1, Daniele Canzani1, Christopher N Woods2

  • 1Department of Chemistry, University of Washington, P.O. Box 351700, Seattle, Washington 98195-1700, United States.

Journal of Proteome Research
|July 5, 2024
PubMed
Summary
This summary is machine-generated.

Genetically incorporating photoreactive amino acids like benzoylphenylalanine (BPA) allows for novel protein crosslinking. A new workflow significantly improves the identification and visualization of these residue-level interactions, aiding structural characterization.

Keywords:
crosslinkingdisorderphoto-crosslinkingprotein structuresmall heat shock proteins

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Area of Science:

  • Biochemistry
  • Structural Biology
  • Proteomics

Background:

  • Conventional crosslinking mass spectrometry is limited by reagent accessibility and reactivity.
  • Genetically incorporated photoreactive amino acids offer broader site targeting and interaction partner reactivity.
  • Identifying crosslinks from photoreactive amino acids presents unique challenges.

Purpose of the Study:

  • To develop and characterize a workflow for identifying and visualizing residue-level protein interactions using genetically incorporated benzoylphenylalanine (BPA).
  • To apply this workflow to an intrinsically disordered region of human HSPB5.
  • To improve the efficiency and precision of crosslink identification compared to existing methods.

Main Methods:

  • Genetically incorporating the photoreactive amino acid benzoylphenylalanine (BPA) into specific sites of the human protein HSPB5.
  • Utilizing a novel workflow for the identification and visualization of BPA-originating crosslinks.
  • Mass spectrometry-based analysis of crosslinked peptides.

Main Results:

  • The workflow routinely identifies 30 to 300 crosslinked peptide spectral matches.
  • This represents a tenfold increase in identified crosslinks compared to existing tools for residue-level BPA crosslink identification.
  • Identified crosslinks are assigned with high precision (one to two residues), supported by reproducible results across replicate analyses.

Conclusions:

  • The developed workflow effectively identifies and visualizes residue-level interactions from genetically incorporated BPA.
  • This method significantly enhances the identification of crosslinks, aiding in the structural characterization of proteins.
  • The workflow is anticipated to facilitate the broader application of photoreactive amino acids for studying challenging protein structures.