Chemical Methods for Encoding and Decoding of Posttranslational Modifications

Abstract

A large array of posttranslational modifications can dramatically change the properties of proteins and influence different aspects of their biological function such as enzymatic activity, binding interactions, and proteostasis. Despite the significant knowledge that has been gained about the function of posttranslational modifications using traditional biological techniques, the analysis of the site-specific effects of a particular modification, the identification of the full complement of modified proteins in the proteome, and the detection of new types of modifications remains challenging. Over the years, chemical methods have contributed significantly in both of these areas of research. This review highlights several posttranslational modifications where chemistry-based approaches have made significant contributions to our ability to both prepare homogeneously modified proteins and identify and characterize particular modifications in complex biological settings. As the number and chemical diversity of documented posttranslational modifications continues to rise, we believe that chemical strategies will be essential to advance the field in years to come.

Figure 1. Encoding and decoding posttranslational modifications (PTMs)

This review covers the different methods available in the chemical toolbox for either the preparation of site-specifically modified proteins (encoding) for subsequent biological experiments or the visualization and identification (decoding) of modifications from living systems and complex protein mixtures.

Figure 2. Encoding protein phosphorylation

(A) A family of ~500 kinases will transfer a phosphate group to certain amino acid side-chains, including serine, threonine, tyrosine, and histidine. (B) Proteins can be synthesized using native chemical ligation (NCL). NCL involves the specific reaction of C-terminal thioesters and N-terminal cysteine residues to form native amide bonds. (C) Recombinant protein thioesters for use in NCL reactions can be created using proteins termed inteins, which catalyze the formation of a branched protein thioester that can be intercepted with exogenous thiols. (D) Unnatural amino acids can be site-specifically incorporated into proteins by using a combination of a tRNA synthetase enzyme that will charge an amber suppressor tRNA with an unnatural amino acid and a corresponding amber stop codon in mRNA.

Figure 3. Decoding protein phosphorylation

(A) Development of analog-sensitive kinases using a bump-hole strategy. Wild-type kinases are incapable of using the “bumped” ATP analog N⁶-benzyl ATP. However, mutation of the kinase in its active site creates a “hole” that will allow N⁶-benzyl ATP to function as a substrate. (B) Identification of kinase substrates using analog-sensitive kinases. A gatekeeper mutant kinase of interest is first incubated with cell lysate and N⁶-benzyl-ATPɣS, resulting in selective thiophosphorylation of that kinase’s substrates. The resulting thiophosphate is then alkylated to generate a p-nitro-benzyl group that is recognized by a specific antibody for visualization or enrichment. (C) Linking a known phosphorylated substrate with the kinase responsible using cross-linking. An ATP based cross-linker is first incubated with a complex mixture of kinases in a cell lysate, transferring a Michael acceptor to the conserved, catalytic lysine residue. Then a substrate peptide bearing a cysteine residue at the known site of phosphorylation is added, yielding a covalent cross-link between the substrate and kinase of interest.

Figure 4. Protein glycosylation

(A) O-linked glycopeptides are typically synthesized through the solution-phase preparation of an Fmoc-protected amino-acid cassette that can be used directly in solid phase peptide synthesis. (B) N-linked glycopeptides have been prepared using the cassette approach but can alternatively synthesized after peptide synthesis through the coupling of an aspartic acid residue to a glycosyl-amine or amine equivalent. (C) Enzymatic installation of large N-linked glycans onto peptides and proteins with transglycosylation reactions. Under certain reaction conditions, some endoglycosidases will use isolated or synthesized glycans as substrates and transfer them onto single N-acetyl-glucosamine residues on peptides or proteins. (D) Metabolic chemical reporters of glycosylation. Living cells are treated with analogs of monosaccharides containing bioorthogonal functionality (e.g., an alkyne). These reporters are metabolized by the cell and installed onto proteins. Bioorthogonal reactions can then be performed for the installation of visualization or affinity tags. (E) Chemoenzymatic detection of O-GlcNAc modifications. Endogenous O-GlcNAc modifications in a cell lysate can be enzymatically modified with a GalNAz residue, followed by the installation of tags using bioorthogonal chemistry.

Figure 5. Ubiquitination

(A) Ubiquitination is the addition of the small protein ubiquitin to protein side chains, most often lysine, resulting in an isopeptide bond. This first modification event can then be polymerized in various ways to form polyubiquitin chains. (B) Synthesis of ubiquitinated histone H2B using a photo-cleavable auxiliary. Using an NCL reaction ubiquitin is first installed onto a synthetic peptide through a lysine residue bearing the auxiliary. Photolysis then both removes the auxiliary and reveals the N-terminal cysteine residue that can be used in subsequent NCL reactions. (C) Ubiquitination of proteins using a δ-mercapto-lysine residue. The δ-mercapto-lysine residue is first incorporated into a peptide using solid phase peptide synthesis, where it can then undergo an NLC reaction with a ubiquitin thioester. The δ-thiol group is then removed by chemical desulfurization. (D) A δ-mercapto-lysine residue can be site specifically installed into recombinant proteins using unnatural amino acid mutagenesis. (E) Examples of isopeptide linkages that have been used for the installation of ubiquitin.

Figure 6. Lipidation

(A) Proteins can be modified by several types of lipids, including myristoylation at the N-termini and palmitoylation and prenylation at cysteine residues. (B) Examples of lipid metabolic chemical reporters for the visualization and identification of lipidated proteins and lipid-dependent protein-protein interactions. (C) Acyl-biotin exchange for the analysis of palmitoylation. Free cysteine residues are first capped by incubation of cell lysates with N-ethylmaleimide. Any palmitate thioesters are then cleaved using hydroxylamine and the resulting free thiols can be reacted with a variety of electrophilic tags.

Figure 7. Acetylation

(A) The most common form of protein acetylation is the dynamic modification of lysine side chains. (B) Acetylated lysine residues can be incorporated into recombinant proteins using unnatural amino acid mutagenesis. (C) Analogs of lysine acetylation can be installed onto cysteine residues using alkylation chemistry, resulting in stable thiocarbamate structures. (D) Chemical reporters of acetylation, malonylation, and aspirin-dependent acetylation.

Figure 8. Methylation and ADP-ribosylation

(A) A variety of methylation marks can be dynamically installed onto both lysine and arginine side chains. (B) Mono-methylation can be incorporated into recombinant proteins using unnatural amino acid mutagenesis followed by acid-based deprotection. (C) Mono-, di-, and tri-methylated lysine analogs can be generated by alkylation of cysteine residues with the appropriate ethyl-amino electrophile. (D) Using a bump-hole approach, the alkyne-bearing SAM analog will be transferred by engineered methyltransferases, enabling the identification of transferase-specific substrates. (E) ADP-ribose can be enzymatically added to a variety of protein side chains and then subsequently polymerized to form long poly-ADP-ribose chains. (F) ADP-ribose analogs can be installed onto peptides after solid phase peptide synthesis by taking advantage of oxime chemistry. (G) Examples of ADP-ribosylation reporters for use in cell lysates.