Se-Glargine: Chemical Synthesis of a Basal Insulin Analogue Stabilized by an Internal Diselenide Bridge.

Insulin has long provided a model for studies of protein folding and stability, enabling enhanced treatment of diabetes mellitus via analogue design. We describe the chemical synthesis of a basal insulin analogue stabilized by substitution of an internal cystine (A6-A11) by a diselenide bridge. The studies focused on insulin glargine (formulated as Lantus® and Toujeo®; Sanofi). Prepared at pH 4 in the presence of zinc ions, glargine exhibits a shifted isoelectric point due to a basic B chain extension (ArgB31 -ArgB32 ). Subcutaneous injection leads to pH-dependent precipitation of a long-lived depot. Pairwise substitution of CysA6 and CysA11 by selenocysteine was effected by solid-phase peptide synthesis; the modified A chain also contained substitution of AsnA21 by Gly, circumventing acid-catalyzed deamidation. Although chain combination of native glargine yielded negligible product, in accordance with previous synthetic studies, the pairwise selenocysteine substitution partially rescued this reaction: substantial product was obtained through repeated combination, yielding a stabilized insulin analogue. This strategy thus exploited both (a) the unique redox properties of selenocysteine in protein folding and (b) favorable packing of an internal diselenide bridge in the native state, once achieved. Such rational optimization of protein folding and stability may be generalizable to diverse disulfide-stabilized proteins of therapeutic interest.

Primordial emergence of a nucleic acid-binding protein via phase separation and statistical ornithine-to-arginine conversion.

De novo emergence demands a transition from disordered polypeptides into structured proteins with well-defined functions. However, can polypeptides confer functions of evolutionary relevance, and how might such polypeptides evolve into modern proteins? The earliest proteins present an even greater challenge, as they were likely based on abiotic, spontaneously synthesized amino acids. Here we asked whether a primordial function, such as nucleic acid binding, could emerge with ornithine, a basic amino acid that forms abiotically yet is absent in modern-day proteins. We combined ancestral sequence reconstruction and empiric deconstruction to unravel a gradual evolutionary trajectory leading from a polypeptide to a ubiquitous nucleic acid-binding protein. Intermediates along this trajectory comprise sequence-duplicated functional proteins built from 10 amino acid types, with ornithine as the only basic amino acid. Ornithine side chains were further modified into arginine by an abiotic chemical reaction, improving both structure and function. Along this trajectory, function evolved from phase separation with RNA (coacervates) to avid and specific double-stranded DNA binding. Our results suggest that phase-separating polypeptides may have been an evolutionary resource for the emergence of early proteins, and that ornithine, together with its postsynthesis modification to arginine, could have been the earliest basic amino acids.

Peptide-RNA Coacervates as a Cradle for the Evolution of Folded Domains.

Peptide-RNA coacervates can result in the concentration and compartmentalization of simple biopolymers. Given their primordial relevance, peptide-RNA coacervates may have also been a key site of early protein evolution. However, the extent to which such coacervates might promote or suppress the exploration of novel peptide conformations is fundamentally unknown. To this end, we used electron paramagnetic resonance spectroscopy (EPR) to characterize the structure and dynamics of an ancient and ubiquitous nucleic acid binding element, the helix-hairpin-helix (HhH) motif, alone and in the presence of RNA, with which it forms coacervates. Double electron-electron resonance (DEER) spectroscopy applied to singly labeled peptides containing one HhH motif revealed the presence of dimers, even in the absence of RNA. Moreover, dimer formation is promoted upon RNA binding and was detectable within peptide-RNA coacervates. DEER measurements of spin-diluted, doubly labeled peptides in solution indicated transient α-helical character. The distance distributions between spin labels in the dimer and the signatures of α-helical folding are consistent with the symmetric (HhH)2-Fold, which is generated upon duplication and fusion of a single HhH motif and traditionally associated with dsDNA binding. These results support the hypothesis that coacervates are a unique testing ground for peptide oligomerization and that phase-separating peptides could have been a resource for the construction of complex protein structures via common evolutionary processes, such as duplication and fusion.

Substitution of an Internal Disulfide Bridge with a Diselenide Enhances both Foldability and Stability of Human Insulin.

Insulin analogues, mainstays in the modern treatment of diabetes mellitus, exemplify the utility of protein engineering in molecular pharmacology. Whereas chemical syntheses of the individual A and B chains were accomplished in the early 1960s, their combination to form native insulin remains inefficient because of competing disulfide pairing and aggregation. To overcome these limitations, we envisioned an alternative approach: pairwise substitution of cysteine residues with selenocysteine (Sec, U). To this end, CysA6 and CysA11 (which form the internal intrachain A6-A11 disulfide bridge) were each replaced with Sec. The A chain[C6U, C11U] variant was prepared by solid-phase peptide synthesis; while sulfitolysis of biosynthetic human insulin provided wild-type B chain-di-S-sulfonate. The presence of selenium atoms at these sites markedly enhanced the rate and fidelity of chain combination, thus solving a long-standing challenge in chemical insulin synthesis. The affinity of the Se-insulin analogue for the lectin-purified insulin receptor was indistinguishable from that of WT-insulin. Remarkably, the thermodynamic stability of the analogue at 25 °C, as inferred from guanidine denaturation studies, was augmented (ΔΔGu ≈0.8 kcal mol-1 ). In accordance with such enhanced stability, reductive unfolding of the Se-insulin analogue and resistance to enzymatic cleavage by Glu-C protease occurred four times more slowly than that of WT-insulin. 2D-NMR and X-ray crystallographic studies demonstrated a native-like three-dimensional structure in which the diselenide bridge was accommodated in the hydrophobic core without steric clash.

Miklós Bodanszky Award Lecture: Selective chalcogen chemistry to study protein science.

In recent decades, chemical protein synthesis and the development of chemoselective reactions-including ligation reactions-have led to significant breakthroughs in protein science. Among them are a better understanding of protein structure-function relationships, the study of protein posttranslational modifications, exploration of protein design, unnatural amino acid incorporation, and the study of therapeutic proteins and protein folding. Chalcogen chemistry, especially that of sulfur and selenium, is quite rich, and we have witnessed continuous progress in this field in recent years. In this short review, we will instead summarize three stories that we have recently presented on chalcogen chemistry and its impact on protein science, which was presented in the Miklós Bodanszky Award Lecture at the 35th European Peptide Society Meeting in Dublin, Ireland, 26 August 2018.