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Related Concept Videos

Protein-protein Interfaces02:04

Protein-protein Interfaces

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Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism's survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a...
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An organism can have thousands of different proteins, and these proteins must cooperate to ensure the health of an organism. Proteins bind to other proteins and form complexes to carry out their functions. Many proteins interact with multiple other proteins creating a complex network of protein interactions.
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Related Experiment Video

Updated: Jul 8, 2025

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study
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Capturing Protein-Protein Interactions with Acidic Amino Acids Reactive Cross-Linkers.

Qing-Qing Liao1,2, Xin Shu1, Wei Sun1

  • 1Zhejiang Provincial Key Laboratory for Cancer Molecular Cell Biology, Life Sciences Institute Cancer Center, Zhejiang University, Hangzhou, Zhejiang, 310058, China.

Small (Weinheim an Der Bergstrasse, Germany)
|December 11, 2023
PubMed
Summary
This summary is machine-generated.

New cross-linkers target acidic residues on protein surfaces, enabling proximity-enhanced reactions at neutral pH. This method efficiently identifies protein-protein interactions, advancing structural biology and proteomics.

Keywords:
acidic amino acid cross‐linkingchemical cross‐linkercross‐linking mass spectrometryp53

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Area of Science:

  • Biochemistry
  • Proteomics
  • Structural Biology

Background:

  • Acidic residues (Aspartic acid and Glutamic acid) are common on protein surfaces, yet chemical cross-linking targeting them is challenging.
  • Existing methods for targeting acidic residues often require high reagent concentrations or exhibit poor structural compatibility.

Purpose of the Study:

  • To develop novel heterobifunctional cross-linkers that react with acidic residues (Asp and Glu) on protein surfaces.
  • To establish a proximity-enhanced cross-linking strategy compatible with neutral pH and room temperature conditions.
  • To expand the utility of cross-linking mass spectrometry (XL-MS) for studying protein-protein interactions involving acidic domains.

Main Methods:

  • Extension of the "plant-and-cast" strategy to create heterobifunctional cross-linkers.
  • Sequential reaction of cross-linkers with Lysine sidechains followed by Aspartic acid and Glutamic acid sidechains.
  • Demonstration of cross-linking efficiency in various model proteins and protein complexes.
  • Application of cross-linking mass spectrometry (XL-MS) to study p53 protein interactions.

Main Results:

  • Developed heterobifunctional cross-linkers that react rapidly with Lys sidechains and then with Asp/Glu sidechains in a proximity-enhanced manner.
  • Cross-linking reactions proceed efficiently at neutral pH and room temperature without the need for coupling reagents.
  • Demonstrated robustness across diverse protein types, from small monomers to large complexes.
  • Identified 87 putative binders of the p53 C-terminal domain using XL-MS, including SARNP, ZRAB2, and WBP11, which regulate p53 target gene expression and splicing.

Conclusions:

  • The novel carboxylate-reactive cross-linkers provide an efficient and structurally compatible method for targeting acidic residues.
  • This approach significantly enhances the capability of XL-MS in mapping protein-protein interactions, particularly those involving acidic domains.
  • The identified p53 interactors highlight the potential of this method for discovering functional protein associations relevant to gene regulation.