Abstract
Genetic code expansion is a powerful tool for the study of protein interactions, as it allows for the site-specific incorporation of a photoreactive group via non-canonical amino acids. Recently, several groups have published bifunctional amino acids that carry a handle for click chemistry in addition to the photo-crosslinker. This allows for the specific labeling of crosslinked proteins and therefore the pulldown of peptides for further analysis. This review describes the properties and advantages of different bifunctional amino acids, and gives an overview about current and future applications.
Highlights
The analysis of interactions between proteins and other biomolecules is a highly important task in both structural biology and systems biology
Genetic code expansion is a powerful tool for the study of protein interactions, as it allows for the site-specific incorporation of a photoreactive group via non-canonical amino acids
Photoreactive non-canonical amino acid (ncAA) introduced via genetic code expansion have been a powerful tool for over 15 years, and were used to find novel protein–protein interactions and to solve the structure of protein complexes [17,23]
Summary
The analysis of interactions between proteins and other biomolecules is a highly important task in both structural biology and systems biology. The formation of a covalent bond between the molecules early in the assay ensures the capture of weaker binding partners Still, this unspecific crosslinking is usually applied after cell lysis, and some interactions might be lost or there may even be false positives introduced [6]. Photoreactive ncAAs introduced via genetic code expansion have been a powerful tool for over 15 years, and were used to find novel protein–protein interactions and to solve the structure of protein complexes [17,23] While this method works well for strong interactions and pure samples, one challenge when using universal photo-crosslinker ncAAs is the sample processing prior to the analysis of the interaction partners [24]. A similar pipeline can be used for structural studies, where the binding partners are usually known, but the binding sites within the molecule are to be determined
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