Disulfide-rich proteins are useful as drugs or tool molecules in biomedical studies, but their synthesis is complicated by the difficulties associated with their folding. Here, we describe a removable glycosylation modification (RGM) strategy that expedites the chemical synthesis of correctly folded proteins with multiple or even interchain disulfide bonds. Our strategy comprises the introduction of simple O-linked β-N-acetylglucosamine (O-GlcNAc) groups at the Ser/Thr sites that effectively improve the folding of disulfide-rich proteins by stabilization of their folding intermediates. After folding, the O-GlcNAc groups can be efficiently removed using O-GlcNAcase (OGA) to afford the correctly folded proteins. Using this strategy, we completed the synthesis of correctly folded hepcidin, an iron-regulating hormone bearing four pairs of disulfide-bonds, and the first total synthesis of correctly folded interleukin-5 (IL-5), a 26 kDa homodimer cytokine responsible for eosinophil growth and differentiation.

The β2-adrenergic receptor (β2AR) is a G-protein-coupled receptor (GPCR) that responds to the hormone adrenaline and is an important drug target in the context of respiratory diseases, including asthma. β2AR function can be regulated by post-translational modifications such as phosphorylation and ubiquitination at the C-terminus, but access to the full-length β2AR with well-defined and homogeneous modification patterns critical for biochemical and biophysical studies remains challenging. Here, we report a practical synthesis of differentially modified, full-length β2AR based on a combined native chemical ligation (NCL) and sortase ligation strategy. An array of homogeneous samples of full-length β2ARs with distinct modification patterns, including a full-length β2AR bearing both monoubiquitination and octaphosphorylation modifications, were successfully prepared for the first time. Using these homogeneously modified full-length β2AR receptors, we found that different phosphorylation patterns mediate different interactions with β-arrestin1 as reflected in different agonist binding affinities. Our experiments also indicated that ubiquitination can further modulate interactions between β2AR and β-arrestin1. Access to full-length β2AR with well-defined and homogeneous modification patterns at the C-terminus opens a door to further in-depth mechanistic studies into the structure and dynamics of β2AR complexes with downstream transducer proteins, including G proteins, arrestins, and GPCR kinases.

Membrane‐associated D‐proteins are an important class of synthetic molecules needed for D‐peptide drug discovery, but their chemical synthesis using canonical ligation methods such as native chemical ligation is often hampered by the poor solubility of their constituent peptide segments. Here, we describe a Backbone‐Installed Split Intein‐Assisted Ligation (BISIAL) method for the synthesis of these proteins, wherein the native L‐forms of the N‐ and C‐intein fragments of the unique consensus‐fast (Cfa) (i.e. L–CfaN and L–CfaC) are separately installed onto the two D‐peptide segments to be ligated via a removable backbone modification. The ligation proceeds smoothly at micromolar (μM) concentrations under strongly chaotropic conditions (8.0 M urea), and the subsequent removal of the backbone modification groups affords the desired D‐proteins without leaving any “ligation scar” on the products. The effectiveness and practicality of the BISIAL method are exemplified by the synthesis of the D‐enantiomers of the extracellular domains of T cell immunoglobulin and ITIM domain (TIGIT) and tropomyosin receptor kinase C (TrkC). The BISIAL method further expands the chemical protein synthesis ligation toolkit and provides practical access to challenging D‐protein targets.