Publikation: Identification of novel functional mini-receptors by combinatorial screening of split-WW domains
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β-Sheet motifs such as the WW domain are increasingly being explored as building blocks for synthetic biological applications. Since the sequence-structure relationships of β-sheet motifs are generally complex compared to the well-studied α-helical coiled coil (CC), other approaches such as combinatorial screening should be included to vary the function of the peptide. In this study, we present a combinatorial approach to identify novel functional mini-proteins based on the WW-domain scaffold, which takes advantage of the successful reconstitution of the fragmented WW domain of hPin1 (hPin1WW) by CC association. Fragmentation of hPin1WW was performed in both loop 1 (CC-hPin1WW-L1) and loop 2 (CC-hPin1WW-L2), and the respective fragments were linked to the strands of an antiparallel heterodimeric CC. Structural analysis by CD and NMR spectroscopy revealed structural reconstitution of the WW-domain scaffold only in CC-hPin1WW-L1, but not in CC-hPin1WW-L2. Furthermore, by using 1H–15N HSQC NMR, fluorescence and CD spectroscopy, we demonstrated that binding properties of fragmented hPin1WW in CC-hPin1WW-L1 were fully restored by CC association. To demonstrate the power of this approach as a combinatorial screening platform, we synthesized a four-by-six library of N- and C-terminal hPin1WW-CC peptide fragments that was screened for a WW domain that preferentially binds to ATP over cAMP, phophocholine, or IP6. Using this screening platform, we identified one WW domain, which specifically binds ATP, and a phosphorylcholine-specific WW-based mini-receptor, both having binding dissociation constants in the lower micromolar range.
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NEITZ, Hermann, Niels Benjamin PAUL, Florian R. HÄGE, Christina LINDNER, Roman GRAEBNER, Michael KOVERMANN, Franziska THOMAS, 2022. Identification of novel functional mini-receptors by combinatorial screening of split-WW domains. In: Chemical Science. Royal Society of Chemistry (RSC). 2022, 13(31), S. 9079-9090. ISSN 2041-6520. eISSN 2041-6539. Verfügbar unter: doi: 10.1039/d2sc01078jBibTex
@article{Neitz2022-08-10Ident-58235, year={2022}, doi={10.1039/d2sc01078j}, title={Identification of novel functional mini-receptors by combinatorial screening of split-WW domains}, number={31}, volume={13}, issn={2041-6520}, journal={Chemical Science}, pages={9079--9090}, author={Neitz, Hermann and Paul, Niels Benjamin and Häge, Florian R. and Lindner, Christina and Graebner, Roman and Kovermann, Michael and Thomas, Franziska} }
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