Functional analysis of protein post-translational modifications using genetic codon expansion

Abstract

Post-translational modifications (PTMs) of proteins not only exponentially increase the diversity of proteoforms, but also contribute to dynamically modulating the localization, stability, activity, and interaction of proteins. Understanding the biological consequences and functions of specific PTMs has been challenging for many reasons, including the dynamic nature of many PTMs and the technical limitations to access homogenously modified proteins. The genetic code expansion technology has emerged to provide unique approaches for studying PTMs. Through site-specific incorporation of unnatural amino acids (UAAs) bearing PTMs or their mimics into proteins, genetic code expansion allows the generation of homogenous proteins with site-specific modifications and atomic resolution both in vitro and in vivo. With this technology, various PTMs and mimics have been precisely introduced into proteins. In this review, we summarize the UAAs and approaches that have been recently developed to site-specifically install PTMs and their mimics into proteins for functional studies of PTMs.

Keywords: bioorthogonal reaction; genetic codon expansion; post-translational modification; unnatural amino acids.

FIGURE 1

The genetic code expansion technology for studying post‐translational modifications (PTMs). (a) Genetic code expansion uses cellular protein translation machinery for site‐specific incorporation of unnatural amino acids (UAAs) into proteins during translation. The genetic code expansion system requires an orthogonal aminoacyl‐tRNA synthetase/tRNA pair together with the gene of interest that carries the UAG amber codon at the target site. The aminoacyl‐tRNA synthetase can selectively catalyze the aminoacylation of its cognate tRNA with the desired UAA to generate the UAA‐charged suppressor tRNA, which recognizes UAG codon and transfers the UAA to the growing peptide chain in the ribosome. As a result, the protein of interest is translated with the UAA at the target site. (b) UAAs containing the native PTMs or mimics are incorporated into the proteins of interest via genetic code expansion. (c) For large PTMs that cannot be directly incorporated, the PTM precursors are incorporated and then converted into the desired PTMs or mimics through chemical or enzymatic transformations

FIGURE 2

Unnatural amino acids that can be genetically incorporated into proteins for studying (a) lysine acetylation, (b) lysine acylations, and (c) acylation defined interactions

FIGURE 3

Unnatural amino acids and approaches based on genetic code expansion for studying lysine ubiquitination. (a) D‐Cys‐ε‐Lys. (b) ε‐N‐tert‐butyloxycarbonyl‐lysine (BocK). (c) ε‐N‐(p‐nitrocarbobenzyloxy)‐δ‐thiol‐lysine. (d) ε‐N‐(2‐propynyloxycarbonyl)‐lysine (Plk). (e) ε‐N‐(tert‐butyloxycarbonyl)‐aminooxy‐lysine. (f) ε‐N‐(2‐azidoacetyl)‐glycyl‐lysine (AzGGK)

FIGURE 4

Unnatural amino acids and approaches based on genetic code expansion for studying (a) lysine monomethylation, (b) lysine dimethylation, and (c) lysine trimethylation

FIGURE 5

Unnatural amino acids and approaches based on genetic code expansion for studying (a) arginine citrullination, (b) serine/threonine phosphorylation, (c) tyrosine phosphorylation, and (d) tyrosine sulfation, nitration, and hydroxylation

FIGURE 6

The genetic code expansion and bioorthogonal reaction approach for studying cysteine S‐fatty‐acylation in live cells. IEDDA: inverse‐electron‐demand Diels–Alder reaction