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Protein-protein Interfaces

Protein-protein Interfaces

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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Updated: Jun 17, 2025

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

Published on: May 5, 2017

Protein-Protein Interface Identification by Site-Specific Photo-Cross-linking/Cleavage in Mammalian Cells.

Kazue Terasawa^1,2, Tatsuro Seike³, Kensaku Sakamoto^4,5

¹Department of Biochemistry, Tokyo Medical and Dental University (TMDU), Bunkyo-ku, Tokyo, Japan.

Current Protocols

|August 6, 2024

Summary

This study introduces a new method for identifying protein-protein interactions using genetic code expansion and site-specific cleavage. The protocol precisely locates cross-linked regions, enhancing the study of protein complexes.

Keywords:

genetic code expansion protein‐protein interface site‐specific cleavage site‐specific crosslinking

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Last Updated: Jun 17, 2025

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Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

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Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Published on: April 1, 2017

Area of Science:

Biochemistry
Molecular Biology
Proteomics

Background:

Protein-protein interactions are crucial for biological processes.
Genetic code expansion allows site-specific photo-cross-linking of proteins.
Identifying cross-linked regions in protein interactions remains a challenge.

Purpose of the Study:

To develop a novel protocol for precise identification of cross-linked regions in protein-protein interactions.
To enable detailed analysis of protein complex structures and binding interfaces.

Main Methods:

Utilizing genetic code expansion to incorporate photo-reactive non-canonical amino acids.
Introducing a site-specific α-hydroxy acid (Nε-allyloxycarbonyl-α-hydroxyl-L-lysine acid, AllocLys-OH) cleavage site.
Employing alkaline treatment for cleavage of the cross-linked complex at the AllocLys-OH residue.

Main Results:

The developed protocol successfully identifies the location of cross-linked sites within target proteins.
Alkaline cleavage at the AllocLys-OH residue precisely determines the proximity of the cross-linked site to the N-terminus or C-terminus.
Iterative introduction of AllocLys-OH effectively narrows down the cross-linked region.

Conclusions:

The combination of site-specific photo-cross-linking and cleavage provides a powerful tool for mapping protein-protein interfaces.
This method facilitates a deeper understanding of protein complex architecture and interaction dynamics.
The protocol is applicable in mammalian cells, offering broad utility in biological research.