Construction of Insulin 18‐mer Nanoassemblies Driven by Coordination to Iron(II) and Zinc(II) Ions at Distinct Sites

Abstract

Controlled self-assembly (SA) of proteins offers the possibility to tune their properties or to create new materials. Herein, we present the synthesis of a modified human insulin (HI) with two distinct metal-ion binding sites, one native, the other abiotic, enabling hierarchical SA through coordination with two different metal ions. Selective attachment of an abiotic 2,2'-bipyridine (bipy) ligand to HI, yielding HI-bipy, enabled Zn(II)-binding hexamers to SA into trimers of hexamers, [[HI-bipy]6]3, driven by octahedral coordination to a Fe(II) ion. The structures were studied in solution by small-angle X-ray scattering and on surfaces with AFM. The abiotic metal ligand had a higher affinity for Fe(II) than Zn(II) ions, enabling control of the hexamer formation with Zn(II) and the formation of trimers of hexamers with Fe(II) ions. This precise control of protein SA to give oligomers of oligomers provides nanoscale structures with potential applications in nanomedicine.

The Poly-Histidine Tag H6 Mediates Structural and Functional Properties of Disintegrating, Protein-Releasing Inclusion Bodies

“…The capability of divalent cations to mediate protein–protein interactions through His-rich domains has been proved in a very different set of contexts [ 21 ], including the in vivo formation of amyloidal structures [ 17 , 20 , 59 , 61 , 64 , 65 , 66 , 67 ]. In vitro, H6 and other related peptides have proved useful for the controlled assembly of pure proteins into a set of amyloidal structures with increasing complexity, from nano- to microscales and with clinical applicability [ 42 , 49 , 68 , 69 , 70 , 71 , 72 ]. The role of polyhistidines in protein aggregation as bacterial IBs had not been so far explored, even those materials are promising as unconventional drug delivery systems [ 23 , 24 ].…”

Enhanced hexamerization of insulin via assembly pathway rerouting revealed by single particle studies

“…ATTO-655-NHS ester (1.0 mg, 0.00122 mmol, 1.0 equivalent) was dissolved in DMF (0.3 mL) and added dropwise over 5 minutes to the stirred solution of insulin aspart, whereafter and allowed the reaction mixture to stir for 15.0 min. The reaction was monitored by LCMS 26,51,52 . Then the reaction mixture was diluted with H 2 O (2.0 mL) and the pH was adjusted to pH 7.8.…”

Half‐Life Extension of Biopharmaceuticals using Chemical Methods: Alternatives to PEGylation

“…It forms higher‐ordered structures, combining native Zn 2+ complexation with abiotic Fe 2+ binding. Insulin 18‐mers were observed by small angle X‐ray scattering in solution, while 54‐mers were observed on surfaces by atomic force microscopy (AFM) . This is a significant step toward construction of novel peptide and protein drugs, which change their oligomeric state in response to metal ion concentration.…”