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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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Protein-Protein Interactions: Surface Plasmon Resonance.

Badreddine Douzi1

  • 1Laboratoire d'Ingénierie des Systèmes Macromoléculaires (LISM, UMR 7255), Institut de Microbiologie de la Méditerranée (IMM), Aix-Marseille Université-Centre National de la Recherche Scientifique (CNRS), 31 Chemin Joseph Aiguier, 13402, Marseille Cedex 20, France. bdouzi@imm.cnrs.fr.

Methods in Molecular Biology (Clifton, N.J.)
|July 2, 2017
PubMed
Summary

Surface Plasmon Resonance (SPR) is a label-free technique to study protein-protein interactions in real-time. This guide details SPR methods for analyzing protein complexes, their binding affinities, and kinetics, particularly for bacterial secretion systems.

Keywords:
AffinityAnalyteBIAcore T200KineticsLigandProtein–protein interactionSurface Plasmon Resonance

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Area of Science:

  • Biophysics
  • Biochemistry
  • Molecular Biology

Background:

  • Protein-protein interactions are crucial for cellular functions, including the assembly and operation of complex molecular machines like bacterial secretion systems.
  • Studying the dynamics, affinities, and kinetics of these interactions is essential for understanding biological processes.
  • Traditional methods often struggle with dynamic complexes, necessitating advanced biophysical techniques.

Purpose of the Study:

  • To provide a comprehensive guide for setting up Surface Plasmon Resonance (SPR) experiments.
  • To detail protocols for identifying protein complexes and quantifying their binding characteristics (affinity and kinetics).
  • To highlight the application of SPR in studying dynamic protein interactions within bacterial secretion systems.

Main Methods:

  • Immobilization of one binding partner (ligand) onto a sensor surface.
  • Injection of the second binding partner (analyte) over the immobilized ligand.
  • Real-time monitoring of binding events via changes in refractive index near the sensor surface.
  • Detailed protocols for amine coupling, analyte binding analysis, affinity/kinetic measurements, and data analysis.

Main Results:

  • SPR enables label-free, real-time measurement of binding affinities and kinetics for protein complexes.
  • The technique is highly effective for studying dynamic and transient interactions, such as those found in secretion systems.
  • SPR can be used for initial screening of interacting partners or validation of interactions identified by other methods.

Conclusions:

  • Surface Plasmon Resonance is a powerful and versatile tool for dissecting protein-protein interactions in various biological contexts.
  • The detailed protocols presented facilitate the application of SPR for in-depth analysis of complex biological systems.
  • Understanding binding affinities and kinetics through SPR is fundamental to elucidating the functional mechanisms of protein machineries.