Linker Helps Detect Protein Partners

Protein-protein interactions play important roles in normal body processes and in disease. Scientists often lock interacting proteins together with cross-linkers to better understand these interactions—information that can help lead to drugs that block or promote the protein-protein interplay. Now, a research team in China has developed a cleavable photo-cross-linking agent that can be genetically engineered into proteins to help study the interactions in living cells.

Cross-linking ensures that protein partners stay together for analysis, especially when interactions are weak or transient. But cross-linking techniques generally do not separate or label protein partners before analysis. The lack of separation or labeling complicates protein identification and can cause the techniques to detect invalid interactions and fail to recognize valid ones.

Peng R. Chen of Peking University and coworkers devised a technique that separates cross-linked interacting proteins prior to analysis and adds a chemical handle that aids detection (J. Am. Chem. Soc. 2014, DOI: 10.1021/ja504371w).

The technique begins with a known “bait protein,” which can interact with “prey proteins.” Chen and coworkers engineer a selenium-based amino acid into the bait protein. Photoactivating the amino acid links bait and prey. And oxidatively cleaving it later separates the partners and leaves the selenium group on the prey.

The selenium group, now oxidized to a selenenic acid, can then be tagged or labeled to facilitate identification of the prey. The researchers used the technique to profile proteins that bind with a bacterial chaperone protein under acidic “stress” conditions.

“Difficulties in isolating cross-linked proteins have been a big impediment to realizing the full power of the photo-cross-linking-MS approach,” comments protein-protein interactions specialist Jennifer Kohler of the University of Texas Southwestern Medical Center, Dallas. “This paper offers an innovative approach to solving this key methodological gap. If selenium cleavage and labeling prove to be robust and reliable in biological lysates, this will be a significant enabling step for cell-based photo-cross-linking.”