Controlling the fractal dimension in self-assembly of terpyridine modified insulin by Fe2+ and Eu3+ to direct in vivo effects

• Published in: Nanoscale 2021-05, Vol.13 (18), p.8467-8473
• Main Authors: Mishra, Narendra Kumar, Østergaard, Mads, Midtgaard, Søren Roi, Strindberg, Sophie S, Winkler, Stefan, Wu, Shunliang, Thomas Just Sørensen, Hassenkam, Tue, Poulsen, Jens-Christian N, Leila Lo Leggio, Hanne Mørck Nielsen, Arleth, Lise, Christensen, Niels Johan, Thulstrup, Peter W, Jensen, Knud J
• Format: Article
• Language: English
• Subjects: Assemblies; Binding; Carboxylic acids; Europium; Ferrous ions; Fluorescence; Fractal geometry; Fractal models; Fractals; Insulin; Metal ions; Oligomerization; Photon correlation spectroscopy; Self-assembly; Small angle X ray scattering; Water chemistry
• ISSN: 2040-3364; 2040-3372
• DOI: 10.1039/d1nr00414j

Metal ion-induced self-assembly (SA) of proteins into higher-order structures can provide new, dynamic nano-assemblies. Here, the synthesis and characterization of a human insulin (HI) analog modified at LysB29 with the tridentate chelator 2,2′:6′,2′′-terpyridine (Tpy) is described. SA of this new insulin analog (LysB29Tpy-HI) in the presence of the metal ions Fe2+ and Eu3+ at different concentrations was studied in solution by fluorescence luminescence and CD spectroscopy, dynamic light scattering, and small-angle X-ray scattering, while surface assembly was probed by AFM. Unique oligomerization was observed in solution, as Fe2+ yielded small magenta-colored discrete non-native assemblies, while Eu3+ caused the formation of large fractal assemblies. Binding of both metal ions to Tpy was demonstrated spectroscopically, and emission lifetime experiments revealed a distinct Eu3+ coordination geometry that included two water molecules. SAXS suggested that LysB29Tpy-HI with Fe2+ oligomerized to a discrete, roughly octameric species, while LysB29Tpy-HI with Eu3+ gave very large assemblies that could be modelled as fractals. The fractal dimensionality increased with the Eu3+ concentration. We propose that this is a consequence of Eu3+ binding to both Tpy and to free carboxylic acid groups on the insulin surface. LysB29Tpy-HI maintained insulin receptor affinity, and showed extended blood glucose lowering and plasma concentration after subcutaneous injection in rats. The combination of metal ion directed SA and native SA provides control of nano-scale fractal dimensionality and points towards use in therapeutics.