The spectrum of building block conformers sustains the biophysical properties of clinically-oriented self-assembling protein nanoparticles

Abstract

Histidine-rich peptides confer self-assembling properties to recombinant proteins through the supramolecular coordination with divalent cations. This fact allows the cost-effective, large-scale generation of microscopic and macroscopic protein materials with intriguing biomedical properties. Among such materials, resulting from the simple bioproduction of protein building blocks, homomeric nanoparticles are of special value as multivalent interactors and drug carriers. Interestingly, we have here identified that the assembly of a given His-tagged protein might render distinguishable categories of self-assembling protein nanoparticles. This fact has been scrutinized through the nanobody-containing fusion proteins EM1-GFP-H6 and A3C8-GFP-H6, whose biosynthesis results in two distinguishable populations of building blocks. In one of them, the assembling and disassembling is controllable by cations. However, a second population immediately self-assembles upon purification through a non-regulatable pathway, rendering larger nanoparticles with specific biological properties. The structural analyses of both model proteins and nanoparticles revealed important conformational variability in the building blocks. This fact renders different structural and functional categories of the final soft materials resulting from the participation of energetically unstable intermediates in the oligomerization process. These data illustrate the complexity of the Hismediated protein assembling in recombinant proteins but they also offer clues for a better design and refinement of protein-based nanomedicines, which, resulting from biological fabrication, show an architectonic flexibility unusual among biomaterials.

Recombinant Proteins for Assembling as Nano- and Micro-Scale Materials for Drug Delivery: A Host Comparative Overview

“…Various engineering approaches allow the production of p and proteins that can self-assemble under the desired conditions. Apart from ratio tein modification towards stabilization [16], the use of histidine (His)-rich segm clustering tags enables coordination and molecular cross-linking with divalent from media [17,18], including those such as Ca 2+ , Mn 2+ , and Zn 2+ that abound in bi tissues [6]. The His-based approach to protein assembly is universal, suited for any type [6,19,20], and useful for generating nanoscale or more complex microscale m depending on the molar ratio between cations and His residues [21] (Figure 1A) fore, tagging with the hexahistidine H6 or similar His-rich peptides is a simple ap that enables proteins of particular biomedical interest to be assembled as regula mers [6,17].…”

Biofabrication of Self-Assembling Covalent Protein Nanoparticles through Histidine-Templated Cysteine Coupling

“…Avoiding the addition of external cations would make the biofabrication process and its scaling-up easier and still greener while minimizing the potential toxicity of gluing ions in specific contexts. Also, the cation-mediated oligomerization is influenced by the conformational status and variability of the His-tagged protein, which cannot be controlled within the cell factory . While some His-tagged proteins self-assemble, supported by the cation content of the media, others need the external supply of such divalent cations or are produced as a split of distinguishable conformer populations …”

“…Also, the cation-mediated oligomerization is influenced by the conformational status and variability of the His-tagged protein, which cannot be controlled within the cell factory . While some His-tagged proteins self-assemble, supported by the cation content of the media, others need the external supply of such divalent cations or are produced as a split of distinguishable conformer populations …”

“…Also, the cationmediated oligomerization is influenced by the conformational status and variability of the His-tagged protein, which cannot be controlled within the cell factory. 38 While some His-tagged proteins self-assemble, supported by the cation content of the media, others need the external supply of such divalent cations or are produced as a split of distinguishable conformer populations. 38 In this context, we envisaged the possibility of generating a linker-free protein-based assembling system with intrinsic reactivity, regardless of an additional source of external cations for histidine coordination.…”

“…38 While some His-tagged proteins self-assemble, supported by the cation content of the media, others need the external supply of such divalent cations or are produced as a split of distinguishable conformer populations. 38 In this context, we envisaged the possibility of generating a linker-free protein-based assembling system with intrinsic reactivity, regardless of an additional source of external cations for histidine coordination. Interestingly, a hybrid, Cyscontaining H6-derivative peptide (H3C) designed to promote covalent cross-interactivity confers, to tagged proteins, robust self-assembling through intersected cysteine residues into cross-linker-free and structurally stable nanoparticles.…”