De novo design and synthesis of EHE miniproteins that contain unnatural β-amino acid building blocks Natalia Miodowska , Anna Szczepańska, Łukasz Berlicki Wrocław University of Science and Technology, Poland The design of miniproteins has drawn much attention in recent years due to their unique structural features, making them the ideal scaffolds for the introduction of functional domains, such as those involved in the inhibition of protein-protein interactions [1,2] and peptide-based catalysis [3,4]. Initially, the design of new miniproteins was based on the redesign of naturally occurring proteins or repeating patterns composed of polar or hydrophobic residues that drive the folding by creating specific secondary subunits. Then, the field of de novo peptides was overtaken by the rational design approach in which biochemical and physicochemical laws, as well as empirical data, play a crucial role. Nowadays, the significance of computational methods which rely on fragment-based design, has greatly increased [5,6]. In our research, we successfully designed conformationally stable miniproteins that fold cooperatively using both computational methods and rational approach. The synthesis of the series of peptides with an EHE topology (i.e. extended-helix-extended), composed of a two-stranded β-sheet and a helix, has shown that they can maintain the stable tertiary structure in an absence of cross-links or a binding metal. Instead, they are stabilised by the packing of the hydrophobic core and the presence of electron-rich Trp regions, which provide additional stabilisation by π-cation interactions. Since the synthetic β-amino acids have been proven to be of a great importance in the formation of peptides with a well-defined structure [7], trans -( 1S,2S )-aminocyclopentanecarboxylic acid ( trans - ACPC) and self-prepared trans -( 3S,4R )-1- N -Boc-4-Fmoc-aminopyrrolidine-3-carboxylic acid ( trans -APC) units were incorporated into the helix creating the αβααβαααβmotif, and α-residues were selected using the Rosetta FastDesign protocol [8] in a way to optimise the packing of the hydrophobic core. The synthesised structures were evaluated by circular dichroism and nanoDSF measurements. All of the obtained miniproteins indicated cooperative folding and good conformational stability, whilst some of them reached estimated melting points exceeding 90 ° C, making them excellent scaffolds of choice. In order to improve physicochemical properties of the peptides, we have decided to replace particular trans -ACPC residues with the corresponding trans -APC units. References 1. Cao et al.: De novo design of picomolar SARS-CoV-2 miniprotein inhibitors, Science (2020) 370, 426–431 S. Haase, K.J. 2. Peterson-Kaufman, S.K. Lan Levengood, J.W. Checco, W.L. Murphy, S.H. Gellman: Extending foldamer design beyond α-helix mimicry: α/β-peptide inhibitors of vascular endothelial growth factor signaling, J. Am. Chem. Soc. (2012) 134, 7652-7655C. 3. Girvin, M.K. Andrews, X. Liu, S.H. Gellman: Foldamer-templated catalysis of macrocycle formation, Science (2019) 366, 1528-1531C. 4. Girvin, S.H. Gellman: Foldamer catalysis, J. Am. Chem. Soc. (2020) 142, 41, 17211–17223V. Korendovych, W.F. DeGrado: De novo protein design, a retrospective, Q. Rev. Biophys. (2020) 53, e3, 1-33 N. Woolfson: A Brief History of De Novo Protein Design: Minimal, Rational, and Computational, J. Mol. Biol. (2021) 433, 20, 167160H. 5. Gellman: Foldamers: A Manifesto, Acc. Chem. Res. (1998) 31, 173-180 Chevalier et al.: Massively parallel de novo protein design for targeted therapeutics, Nature (2017) 550, 74-79
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