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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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Proteins are one of the most abundant organic molecules in living systems and have the most diverse range of functions of all macromolecules. Proteins may be structural, regulatory, contractile, or protective. They may serve in transport, storage, or membranes; or they may be toxins or enzymes. Their structures, like their functions, vary greatly. They are all, however, amino acid polymers arranged in a linear sequence.
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Yeasts are single-celled organisms, but unlike bacteria, they are eukaryotes (cells with a nucleus). Cell signaling in yeast is similar to signaling in other eukaryotic cells. A ligand, such as a protein or a small molecule released from a yeast cell, attaches to a receptor on the cell surface. The binding stimulates second-messenger kinases to activate or inactivate transcription factors that further regulate gene expression. Many of the yeast intracellular signaling cascades have similar...
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Groups of proteins may form a complex where each protein in this complex has a different role in the overall execution of the complex’s function. Often some of the proteins in the complex can be replaced by a closely related variant to give a complex that contains many of the same components yet is functionally distinct.
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A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions
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Identification of Protein-Protein Interactions Using Pool-Array-Based Yeast Two-Hybrid Screening.

Thomas Lathouwers1, Jeroen Wagemans1, Rob Lavigne2

  • 1Laboratory of Gene Technology, KU Leuven, Leuven, Belgium.

Methods in Molecular Biology (Clifton, N.J.)
|June 2, 2018
PubMed
Summary

This study details an array-based yeast two-hybrid (Y2H) screening method using a pooling strategy to efficiently identify protein interaction networks. The protocol covers bait transformation, mating, and result evaluation for comprehensive network analysis.

Keywords:
ArraysPoolingProtein–protein interactionsYeast two-hybridα-Galactosidase assay

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Protein-protein interactions are fundamental to cellular processes.
  • Yeast two-hybrid (Y2H) is a powerful technique for studying these interactions.
  • Array-based Y2H with pooling enhances screening efficiency.

Purpose of the Study:

  • To provide a detailed protocol for array-based Gal4p Y2H screening.
  • To demonstrate the utility of a pooling strategy for improved experimental efficacy.
  • To outline methods for confirming interactions and quantifying interaction strengths.

Main Methods:

  • Array-based screening using Gal4p yeast two-hybrid system.
  • Implementation of a pooling strategy for baits and preys.
  • Detailed steps including bait transformation, autoactivation testing, yeast mating, and result evaluation.
  • One-on-one confirmation and quantitative alpha-galactosidase assays.

Main Results:

  • The described protocol enables the identification of protein interaction networks.
  • Pooling strategy enhances the efficiency of large-scale Y2H screening.
  • Confirmation and quantitative assays allow for robust validation of interactions.

Conclusions:

  • Array-based Y2H screening with pooling is an effective method for mapping protein interaction networks.
  • The protocol provides a comprehensive guide for researchers to implement this technique.
  • This approach facilitates a deeper understanding of molecular mechanisms through interaction analysis.