Genetic Encoding of Phosphorylated Amino Acids into Proteins

Reversible phosphorylation is a fundamental mechanism
for controlling
protein function. Despite the critical roles phosphorylated proteins
play in physiology and disease, our ability to study individual phospho-proteoforms
has been hindered by a lack of versatile methods to efficiently generate
homogeneous proteins with site-specific phosphoamino acids or with
functional mimics that are resistant to phosphatases. Genetic code
expansion (GCE) is emerging as a transformative approach to tackle
this challenge, allowing direct incorporation of phosphoamino acids
into proteins during translation in response to amber stop codons.
This genetic programming of phospho-protein synthesis eliminates the
reliance on kinase-based or chemical semisynthesis approaches, making
it broadly applicable to diverse phospho-proteoforms. In this comprehensive
review, we provide a brief introduction to GCE and trace the development
of existing GCE technologies for installing phosphoserine, phosphothreonine,
phosphotyrosine, and their mimics, discussing both their advantages
as well as their limitations. While some of the technologies are still
early in their development, others are already robust enough to greatly
expand the range of biologically relevant questions that can be addressed.
We highlight new discoveries enabled by these GCE approaches, provide
practical considerations for the application of technologies by non-GCE
experts, and also identify avenues ripe for further development.