Antigen-Detected NMR for Minimal Epitope Engineering and Structure-Guided Selection of a Na(V)1.7-Selective Nanobody
- Author(s)
- Liu, J; Chen, W; Cristofori-Armstrong, B; Crawford, T; Chen, KE; Xie, P; Chan, RWB; Zhu, Y; Golder, M; Pereira Schmidt, A; Naughton, JD; Condon, ND; Andersson, Å; Dehkhoda, F; McMahon, KL; Klasfauseweh, T; Thapa, A; Tran, H; Tran, P; Jami, S; Ragnarsson, L; Furness, SGB; Deuis, JR; Collins, BM; Tham, WH; Prasadam, I; Vetter, I; Mobli, M;
- Journal Title
- Advanced Science
- Publication Type
- Sep 6
- Abstract
- Selective molecular recognition of membrane proteins is challenging because they contain few solvent-exposed extracellular epitopes, which often depend on their native environment for structure, making them difficult to isolate faithfully for binder discovery. Here, we show that antigen-detected NMR is well suited both to characterizing the folding of engineered minimal epitopes from the human voltage-gated sodium channel Na(V)1.7 and to selecting binders that recognize their solvent-exposed surfaces. Isotope labelling of the antigen enables NMR resonance assignment to assess retained local secondary structure, while (15)N titration and zz-exchange mapping provide binding and interface information. Combined with AlphaFold2 complex prediction, this creates a practical method for screening and ranking candidate binders. The approach was further validated by a high-resolution x-ray structure of an antigen-nanobody complex. Applying this workflow identified R4C8, a subtype- and species-selective nanobody whose binding to the extracellular surface of human Na(V)1.7 is supported by zz-exchange mapping, modelling, and cellular recognition, and which has minimal effects on channel gating. R4C8 detected Na(V)1.7 in engineered cell lines and in primary osteoarthritis-derived chondrocytes, providing a useful tool for selective target detection. These results show how antigen-detected NMR can support peptide engineering and structure-guided protein binder selection against minimal epitopes.
- Publisher
- Wiley
- Keywords
- NMR spectroscopy; NaV1.7; chondrocytes; nanobody; protein binder
- Research Division(s)
- Infection and Global Health
- PubMed ID
- 42702812
- Publisher's Version
- https://doi.org/10.1002/advs.77611
- Open Access at Publisher's Site
https://doi.org/10.1002/advs.77611- Terms of Use/Rights Notice
- Refer to copyright notice on published article.
Creation Date: 2026-09-14 08:56:33
Last Modified: 2026-09-14 08:56:54