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α1-FANGs: Protein Ligands Selective for the α-Bungarotoxin Site of the α1-Nicotinic Acetylcholine Receptor

Nichols, Aaron L. and Noridomi, Kaori and Hughes, Christopher R. and Jalali-Yazdi, Farzad and Eaton, J. Brek and Lai, Lan Huong and Advani, Gaurav and Lukas, Ronald J. and Lester, Henry A. and Chen, Lin and Roberts, Richard W. (2018) α1-FANGs: Protein Ligands Selective for the α-Bungarotoxin Site of the α1-Nicotinic Acetylcholine Receptor. ACS Chemical Biology, 13 (9). pp. 2568-2576. ISSN 1554-8929. PMCID PMC8763392. doi:10.1021/acschembio.8b00513.

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[img] PDF (Plot of ΔGunfolding versus inverse temperature of α1-FANG1 and its derivatives (Supplemental Figure 1) and determination of α1-FANG3 Kon and Koff rates via radiolabeled binding assays (Supplemental Figure 2)) - Supplemental Material
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Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that play a central role in neuronal and neuromuscular signal transduction. Here, we have developed FANG ligands, fibronectin antibody-mimetic nicotinic acetylcholine receptor-generated ligands, using mRNA display. We generated a 1 trillion-member primary e10FnIII library to target a stabilized α1 nicotinic subunit (α211). This library yielded 270000 independent potential protein binding ligands. The lead sequence, α1-FANG1, represented 25% of all library sequences, showed the highest-affinity binding, and competed with α-bungarotoxin (α-Btx). To improve this clone, a new library based on α1-FANG1 was subjected to heat, protease, binding, off-rate selective pressures, and point mutations. This resulted in α1-FANG2 and α1-FANG3. These proteins bind α211 with KDvalues of 3.5 nM and 670 pM, respectively, compete with α-Btx, and show improved subunit specificity. α1-FANG3 is thermostable (T_m = 62 °C) with a 6 kcal/mol improvement in folding free energy compared with that of the parent α1-FANG1. α1-FANG3 competes directly with the α-Btx binding site of intact neuromuscular heteropentamers [(α1)_2β1γδ] in mammalian culture-derived cellular membranes and in Xenopus laevis oocytes expressing these nAChRs. This work demonstrates that mRNA display against a monomeric ecto-domain of a pentamer has the capability to select ligands that bind that subunit in both a monomeric and a pentameric context. Overall, our work provides a route to creating a new family of stable, well-behaved proteins that specifically target this important receptor family.

Item Type:Article
Related URLs:
URLURL TypeDescription Information CentralArticle
Nichols, Aaron L.0000-0001-9341-0049
Lester, Henry A.0000-0002-5470-5255
Roberts, Richard W.0000-0002-8587-5097
Additional Information:© 2018 American Chemical Society. Received: June 1, 2018; Accepted: July 30, 2018; Published: July 30, 2018. We would like to thank Caitlin Nichols for her acronym expertise and Dr. Matthew Mulcahy for his digital prowess. Supported by National Institutes of Health Grants R01 AI085583 and R01 DA037161. The authors declare no competing financial interest.
Funding AgencyGrant Number
NIHR01 AI085583
NIHR01 DA037161
Issue or Number:9
PubMed Central ID:PMC8763392
Record Number:CaltechAUTHORS:20180924-141114616
Persistent URL:
Official Citation:α1-FANGs: Protein Ligands Selective for the α-Bungarotoxin Site of the α1-Nicotinic Acetylcholine Receptor. Aaron L. Nichols, Kaori Noridomi, Christopher R. Hughes, Farzad Jalali-Yazdi, J. Brek Eaton, Lan Huong Lai, Gaurav Advani, Ronald J. Lukas, Henry A. Lester, Lin Chen, and Richard W. Roberts. ACS Chemical Biology 2018 13 (9), 2568-2576. DOI: 10.1021/acschembio.8b00513
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89894
Deposited By: Tony Diaz
Deposited On:24 Sep 2018 22:45
Last Modified:19 Jan 2022 18:16

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