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Abstract

The current goal of research in the field of synthetic chemistry is the design and production of peptide and protein structures that function as biological synthesis machines.[1] Initially, the de novo design of peptide catalysts focused on α-helical structural motifs, such as the coiled coil.[2,3] Given the relatively rigid and self-assembling nature of these scaffolds[4,5], scientific efforts are concentrated on the design of smaller single-chain β-sheet motifs, such as the WW domain.[6–9] Due to its properties as a small, independently folding protein motif with 34-40 amino acid residues, as well as its function as a protein interaction module with a flexible binding site, the WW domain was selected as a scaffold for the design of miniaturized proteins in this work.[10]

To establish the WW domain as a scaffold for miniprotein design, an iterative sequence analysis approach was initially employed to identify sequence-structure-stability-relationships, starting from a consensus sequence of 90 native WW domains. In accordance with the identified sequence-structurestability relationships, three highly thermostable WW domain core scaffolds were designed, displaying melting temperatures of 89 and 93 °C in thermal CD denaturation experiments. Multi-dimensional high-resolution NMR experiments of one highly thermostable scaffold peptide demonstrated the typical WW domain structure, comprising three anti-parallel β-sheets and additional structural features that contribute to its high thermostability. As a proof of concept for the design of functional miniproteins, binding motifs of the three different WW domain groups were grafted onto the thermostable WW domain core scaffold to obtain thermostable WW domains with group-specific binding properties. Binding studies on the designed group-specific thermostable WW domains revealed micromolar-to-sub-micromolar binding affinities to their respective ligands. In addition to the introduction of native functions into the thermostable WW domain core scaffold, binding motifs for non-native functions, such as metal- and organophosphate binding, were grafted onto a scaffold peptide. This resulted in the design of de novo thermostable WW domains with micromolar-tosubmicromolar binding affinities to their respective ligands. Following the successful design of an adenosine-triphosphate (ATP)-binding thermostable variant (WW-HS-ATP), further peptide variants were designed with diverse sequence modifications to study the influence on binding affinity to ATP, ADP and AMP. All de novo designed variants, as well as WW-HS-ATP, demonstrated micromolar binding affinities to ATP, ADP and AMP. To ascertain whether one of the ATP-binding peptides exhibits enzymatic activity in terms of phosphatase activity, exploratory studies were conducted, which identified certain candidates that showed minimal phosphatase-like activity. To obtain thermostable WW domain miniproteins with enzymatic activity, further sequence redesign and additional studies are required. In conclusion, this work successfully demonstrated that the establishment of sequence-structure-stability relationships of native WW domains, resulted in the design of highly thermostable WW domain core scaffolds, which could be further functionalized with native and non-native functions, while maintaining thermostable properties. The designed thermostable WW domain scaffolds could serve as a template for future β-sheet miniprotein design with diverse receptor, sensor or catalytic properties.