Antigen‐Detected NMR for Minimal Epitope Engineering and Structure‐Guided Selection of a Na V 1.7‐Selective Nanobody

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.

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Publication Details

Journal
Advanced Science
Published
2026-09-06
DOI
https://doi.org/10.1002/advs.77611
Primary Topic
Monoclonal and Polyclonal Antibodies Research
Type
article
Field-Weighted Citation Impact
0.00
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article

Antigen‐Detected NMR for Minimal Epitope Engineering and Structure‐Guided Selection of a Na V 1.7‐Selective Nanobody

Irina Vetter, Åsa Andersson, Theo Crawford, Brett M. Collins et al.
Advanced Science
Monoclonal and Polyclonal Antibodies Research
article

Antigen‐Detected NMR for Minimal Epitope Engineering and Structure‐Guided Selection of a Na V 1.7‐Selective Nanobody

Irina Vetter, Åsa Andersson, Theo Crawford, Brett M. Collins, Farhad Dehkhoda, Mehdi Mobli, Lotten Ragnarsson, Wai‐Hong Tham, Sina Jami, Sebastian G. B. Furness, Poanna Tran, Tabea Klasfauseweh, Ashvriya Thapa, Mimi Golder, Nicholas D. Condon, Kai‐En Chen, Ben Cristofori‐Armstrong, Jennifer R. Deuis, Peng Xie, Indira Prasadam, Jennifer D. Naughton, Junyu Liu, Wanlin Chen, Yifei Zhu, Anneka Pereira Schmidt, Kirsten L. McMahon, Hue Tran, Rainbow Wing Bo Chan
article en

Abstract

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.

Advanced Science
Australian National University (AU), Queensland University of Technology (AU), The University of Queensland (AU), The University of Melbourne (AU), Walter and Eliza Hall Institute of Medical Research (AU), Monash University (AU)
Life in Land
Openalex Percentile: Top 11%
Monoclonal and Polyclonal Antibodies Research
DOIOpen Access PDFOpenAlex
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