The Journey for the Total Chemical Synthesis of a 53 kDa Protein.

Chemical protein synthesis has been proved as an efficient way to afford medium-sized proteins with high homogeneity in workable quantities for various biochemical, structural, and functional studies. In particular, chemical protein synthesis has enabled access to proteins that are difficult or impossible to prepare by molecular biology approaches, such as those with post-translational modifications and mirror-image proteins. One prominent example is related to ubiquitination, a well-known modification that mediates a variety of cellular processes (e.g., proteasomal degradation). Ubiquitination is considered as a modification that is difficult to introduce into proteins in a test tube to generate ubiquitin (Ub) conjugates with high homogeneity with respect to the chain length and the anchored Lys residue in workable quantities to perform the biochemical and biophysical studies. Chemical protein synthesis has emerged as a powerful approach to prepare Ub conjugates for studies aiming to understand ubiquitination in great detail and decipher its roles in cell processes. Nevertheless, in order to answer more challenging questions in this field, it has been clear that researchers must also prepare Ub conjugates with increased size and complexity. Employing solid-phase peptide synthesis and chemoselective ligation, chemical protein synthesis offers a powerful way to furnish polypeptides composed of 100-200 residues. However, to synthesize larger proteins such as Ub conjugates, longer and more segments are required. This on the other hand leads to difficulties related to solubility, purification, ligation, and late-stage modifications. These challenges have encouraged us to explore more practical synthetic tools to facilitate the synthesis of complex Ub conjugates. In this Account, we summarize the synthetic tools that we have developed to achieve these goals. These include (1) δ-mercaptolysine-mediated isopeptide chemical ligation, (2) chemical synthesis of Ub building blocks, (3) palladium-mediated deprotection of key side chains during protein synthesis, (4) one-pot ligation and desulfurization, and (5) improving the solubility of peptide segments. The developed chemical toolbox has been a key for our successes in the synthesis of diverse and complex Ub conjugates. In this Account, we describe our approaches for generating various Ub conjugates, including (1) the K48 tetra-Ub chain composed of 304 amino acids, (2) the ubiquitinated histones and their analogues made of >200 amino acids, (3) the di-Ub-SUMO-2 hybrid chain composed of 245 amino acids, and (4) the 53 kDa tetra-Ub-α-globin composed of 472 amino acids, which represents the largest protein composed of natural amino acids ever made using chemical protein synthesis. The last target, Flag-Ub-Ub-Ub-Myc-Ub-(HA-α-globin), was prepared in the labeled form where the proximal Ub and distal Ub in the chain were labeled with Myc and Flag tags, respectively, while the α-globin was labeled with the HA tag. Applying the tetra-Ub-α-globin in proteasomal degradation studies assisted us to shed light on the proteolytic signal and the fates of the Ub moieties in the chains. Although these developments have contributed to the synthesis of interesting and challenging targets related to Ub signaling, several other targets may enforce new synthetic challenges. Hence, there is still a need to optimize the current synthetic tools and explore novel synthetic approaches to facilitate this process.