Zhang Xiaorong, Waffo Armel T, Yarman Aysu, Kovács Norbert, Bognár Zsófia, Wollenberger Ulla, El-Sherbiny Ibrahim M, Hassan Rabeay Y A, Bier Frank F, Gyurcsányi Róbert E, Zebger Ingo, Scheller Frieder W
Institute of Biochemistry and Biology, University of Potsdam, Karl-Liebknecht Str. 24-25, 14476 Potsdam, Germany.
Institut für Chemie, PC 14 Technische Universität Berlin, Straße des 17. Juni 135, 10623 Berlin, Germany.
Nanoscale. 2022 Dec 15;14(48):18106-18114. doi: 10.1039/d2nr03898f.
Here we aim to gain a mechanistic understanding of the formation of epitope-imprinted polymer nanofilms using a non-terminal peptide sequence, the peptide GFNCYFP (G485 to P491) of the SARS-CoV-2 receptor binding domain (RBD). This epitope is chemisorbed on the gold surface through the central cysteine 488 followed by the electrosynthesis of a ∼5 nm thick polyscopoletin film around the surface confined templates. The interaction of peptides and the parent RBD and spike protein with the imprinted polyscopoletin nanofilm was followed by electrochemical redox marker gating, surface enhanced infrared absorption spectroscopy and conductive AFM. Because the use of non-terminal epitopes is especially intricate, here we characterize the binding pockets through their interaction with 5 peptides rationally derived from the template sequence, implementing central single amino acid mismatch as well as elongations and truncations at its C- and N- termini. Already a single amino acid mismatch, the central Cys488 substituted by a serine, results in 15-fold lower affinity. Further truncation of the peptides to tetrapeptide (EGFN) and hexapeptide (YFPLQS) results also in a significantly lower affinity. We concluded that the affinity towards the different peptides is mainly determined by the four amino acid motif CYFP present in the sequence of the template peptide. A higher affinity than that for the peptides is found for the parent proteins RBD and spike protein, which seems to be due to out of cavity effects caused by their larger footprint on the nanofilm surface.
在这里，我们旨在通过使用非末端肽序列——严重急性呼吸综合征冠状病毒2（SARS-CoV-2）受体结合域（RBD）的肽GFNCYFP（G485至P491），来深入了解表位印记聚合物纳米膜的形成机制。该表位通过中央半胱氨酸488化学吸附在金表面，随后在表面受限模板周围电合成约5纳米厚的聚司可波letin膜。通过电化学氧化还原标记门控、表面增强红外吸收光谱和导电原子力显微镜，研究了肽以及亲本RBD和刺突蛋白与印记聚司可波letin纳米膜的相互作用。由于使用非末端表位特别复杂，在这里我们通过与从模板序列合理衍生的5种肽的相互作用来表征结合口袋，在其C端和N端实施中央单氨基酸错配以及延伸和截断。仅仅一个氨基酸错配，即中央半胱氨酸488被丝氨酸取代，就会导致亲和力降低15倍。将肽进一步截短为四肽（EGFN）和六肽（YFPLQS）也会导致亲和力显著降低。我们得出结论，对不同肽的亲和力主要由模板肽序列中存在的四氨基酸基序CYFP决定。亲本蛋白RBD和刺突蛋白的亲和力高于肽，这似乎是由于它们在纳米膜表面较大的覆盖面积所引起的腔外效应。
