A photo-cross-linking approach to monitor protein dynamics in living cells

Background: Proteins, which comprise one of the major classes of biomolecules that constitute a cell, interact with other cellular factors during both their biogenesis and functional states. Studying not only static but also transient interactions of proteins is important to understand their physiological roles and regulation mechanisms. However, only a limited number of methods are available to analyze the dynamic behaviors of proteins at the molecular level in a living cell. The site-directed in vivo photo-cross-linking approach is an elegant technique to capture protein interactions with high spatial resolution in a living cell. Scope of review: Here, we review the in vivo photo-cross-linking approach including its recent applications and the potential problems to be considered. We also introduce a new in vivo photo-cross-linking-based technique (PiXie) to study protein dynamics with high spatiotemporal resolution. Major conclusions: In vivo photo-cross-linking enables us to capture weak/transient protein interactions with high spatial resolution, and allows for identification of interacting factors. Moreover, the PiXie approach can be used to monitor rapid folding/assembly processes of proteins in living cells. General significance: In vivo photo-cross-linking is a simple method that has been used to analyze the dynamic interactions of many cellular proteins. Originally developed in Escherichia coli, this system has been extended to studies in various organisms, making it a fundamental technique for investigating dynamic protein interactions in many cellular processes. This article is part of a Special issue entitled Novel major techniques for visualizing `live' protein molecules edited by Dr. Daisuke Kohda.