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.
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Acknowledgements
We are indebted to the Agencia Estatal de Investigación (AEI) and to the Fondo Europeo de Desarrollo Regional (FED-ER) (PID2020-116174RB-I00, AEI/FEDER, UE). The AGAUR (2017SGR-229) and the CIBER-BBN (project NANOPROTHER) are granted to Villaverde A. Vázquez E received support from the AEI (PID2019-105416RB-I00/AEI/10.13039/501100011033 and CIBER-BBN (project NANOREMOTE). We also appreciate the support from the Javna Agencija za Raziskovalno dejavnost Republike Slovenije (ARRS/N4-0046 and ARRS/J4-9322) to de Marco A. Sánchez-García L was supported by a predoctoral fellowship from the AGAUR (2018FI_B2_00051), Voltà-Durán E by a predoctoral fellowship from Ministerio de Ciencia, Innovacion y Universidades (FPU18/04615), Unzueta U was supported by Miguel Servet contract (CP19/00028) from ISCIII co-funded by European Social Fund (ESF investing in your future) and by an ISCIII project (PI20/00400) co-funding FEDER (A way to make Europe). López-Laguna H was supported by a predoctoral fellowship from the AGAUR (2019FI_B00352). Villaverde A received an ICREA ACADEMIA award. Protein production was partially performed by the ICTS “NAN-BIOSIS”, more specifically by the Protein Production Platform of CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN)/IBB, at the UAB (http://www.nanbiosis.es/portfolio/u1-protein-production-platform-ppp/). Cell culture experiments were performed at SCAC facilities in the UAB. Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311. Electron microscopy studies were performed by the Servei de Microscòpia in the UAB.
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Author contributions Voltà-Durán E performed most of the experiments and prepared the figures; Sánchez JM, López-Laguna H and Parladé E performed the structural analyses and prepared the figures; Sánchez-García L designed and produced the nanobody constructs under the supervision of de Marco A; Sánchez-Chardi A analyzed the data; Unzueta U supervised most of the experiments; Vázquez E and Villaverde A conceived and supervised the whole study; Villaverde A prepared the first draft of the manuscript. All authors contributed to the general discussion and approved the manuscript.
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Supplementary information The original data presented in this paper can be found at: https://ddd.uab.cat/record/248706.
Eric Voltà-Durán graduated in biotechnology in 2018 and pursued Master studies in advanced nanoscience and nanotechnology (2019). He is a young, early-stage researcher at the Nanobiotechnology group, a team led by Prof. Villaverde at the Autonomous University of Barcelona (UAB). He is working on his PhD studies, in which he focuses on the design and production of proteins with biomedical interest, specially in the context of targeted therapies.
Antonio Villaverde graduated in biological sciences in 1982 and got his PhD degree in 1985. He has been scientifically formed in Barcelona, Madrid, London, Lausanne and Braunschweig. Since 1987, he has been professor of microbiology at the Universitat Autònoma de Barcelona, Spain, where he got a full professorship in 2002. He leads the Nanobiotechnology group in this university and in the CIBER-BBN, focusing on the design of protein-based materials for biomedical applications. He founded the journal Microbial Cell Factories in 2002, being its Editor-in-Chief for 15 years.
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Voltà-Durán, E., Sánchez, J.M., López-Laguna, H. et al. The spectrum of building block conformers sustains the biophysical properties of clinically-oriented self-assembling protein nanoparticles. Sci. China Mater. 65, 1662–1670 (2022). https://doi.org/10.1007/s40843-021-1914-0
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DOI: https://doi.org/10.1007/s40843-021-1914-0