{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T05:07:04Z","timestamp":1789016824204,"version":"build-2803163510"},"reference-count":36,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T00:00:00Z","timestamp":1788220800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Project of Collaborative Innovation Center of GDAAS","award":["XT202504"],"award-info":[{"award-number":["XT202504"]}]},{"name":"Project of Collaborative Innovation Center of GDAAS","award":["XT202210"],"award-info":[{"award-number":["XT202210"]}]},{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["22307022"],"award-info":[{"award-number":["22307022"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["22307022"],"award-info":[{"award-number":["22307022"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"name":"Youth S&T Talent Support Programme of Guangdong Provincial Association for Science and Technology","award":["SKXRC2025504"],"award-info":[{"award-number":["SKXRC2025504"]}]},{"name":"Special Fund for Scientific Innovation Strategy-Construction of High Level Academy of Agriculture Science","award":["R2023PY-JX024"],"award-info":[{"award-number":["R2023PY-JX024"]}]},{"name":"Special Fund for Scientific Innovation Strategy-Construction of High Level Academy of Agriculture Science","award":["R2021YJ-YB1004"],"award-info":[{"award-number":["R2021YJ-YB1004"]}]},{"name":"Special Fund for Scientific Innovation Strategy-Construction of High Level Academy of Agriculture Science","award":["2025ZQQZ-G19"],"award-info":[{"award-number":["2025ZQQZ-G19"]}]},{"name":"Special Fund for Scientific Innovation Strategy-Construction of High Level Academy of Agriculture Science","award":["2023QZ-NK04"],"award-info":[{"award-number":["2023QZ-NK04"]}]},{"name":"Special Fund for Scientific Innovation Strategy-Construction of High Level Academy of Agriculture Science","award":["GDNKY-ZQQZ-K5"],"award-info":[{"award-number":["GDNKY-ZQQZ-K5"]}]},{"name":"Special Fund for Scientific Innovation Strategy-Construction of High Level Academy of Agriculture Science","award":["NYQS202628"],"award-info":[{"award-number":["NYQS202628"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Aflatoxin B1 (AFB1) is a potent foodborne mycotoxin for which nanobodies offer compact, engineerable detection reagents, yet the molecular basis of their affinity differences remains poorly defined. Here, closely related nanobodies G8, NB28, and NB26 were compared. Fluorescence correlation spectroscopy showed G8 had the lowest dissociation constant for AFB1-BSA, followed by NB28 and NB26. To resolve this affinity separation, we performed triplicate 3 \u03bcs molecular dynamics simulations for each complex. While all complexes remained stable, G8 maintained broader, persistent aromatic and hydrophobic contacts extending into its elongated CDR3, whereas NB28 and NB26 relied on localized contacts centered on PHE47 and TRP101. Per-residue MM\/GBSA decomposition confirmed favorable thermodynamic contributions from these persistent G8 residues. Ultimately, integrating contact occupancy, energy decomposition, and pairwise co-occurrence identified distinct cooperative contact networks (six in G8, versus two in NB28\/NB26). This workflow demonstrates how mapping dynamic, cooperative binding networks rather than static proximity can systematically prioritize candidate residues for engineering high-affinity detection reagents in food-safety surveillance.<\/jats:p>","DOI":"10.3390\/biom16091265","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T16:28:22Z","timestamp":1788280102000},"page":"1265","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Contact Occupancy and Per-Residue Energetic Contributions Distinguish Aflatoxin B1 Nanobodies with Different Binding Affinities"],"prefix":"10.3390","volume":"16","author":[{"given":"Hualian","family":"Mo","sequence":"first","affiliation":[{"name":"State Key Laboratory of Swine and Poultry Breeding Industry, Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"visualization","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"}]},{"given":"Wenxing","family":"Chen","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Swine and Poultry Breeding Industry, Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Zhaoxi","family":"Yang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Swine and Poultry Breeding Industry, Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China"},{"name":"School of Life Sciences, South China Normal University, Guangzhou 510631, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Junjie","family":"Zhou","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Swine and Poultry Breeding Industry, Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Xinze","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Swine and Poultry Breeding Industry, Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Siyuan","family":"Yang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Swine and Poultry Breeding Industry, Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Xiaoman","family":"She","sequence":"additional","affiliation":[{"name":"Guangdong Provincial Key Laboratory of High Technology for Plant Protection, Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"methodology","vocabulary":"credit"}]},{"given":"Wenyang","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Swine and Poultry Breeding Industry, Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"}]},{"given":"Shijuan","family":"Yan","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Swine and Poultry Breeding Industry, Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9885-5642","authenticated-orcid":false,"given":"Shaowen","family":"Wu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Swine and Poultry Breeding Industry, Agro-Biological Gene Research Center, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"methodology","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"102251","DOI":"10.1016\/j.afres.2026.102251","article-title":"Mycotoxin exposure: Risks to human health and detection approaches","volume":"6","author":"Rathore","year":"2026","journal-title":"Appl. Food Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"409","DOI":"10.3920\/WMJ2018.2346","article-title":"Probabilistic dietary based estimation of the burden of aflatoxin-induced hepatocellular carcinoma among adult Malawians","volume":"12","author":"Matumba","year":"2019","journal-title":"World Mycotoxin J."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Wei, Y., Nie, Y., Yan, S., Teng, C., and Wu, S. (2026). Toxic effects and detoxification strategies of aflatoxin B1. Anim. Zoonoses.","DOI":"10.1016\/j.azn.2026.02.006"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"133287","DOI":"10.1016\/j.foodchem.2022.133287","article-title":"Single-step noncompetitive immunocomplex immunoassay for rapid aflatoxin detection","volume":"392","author":"Peltomaa","year":"2022","journal-title":"Food Chem."},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Gonzalez-Sapienza, G., Rossotti, M.A., and Tabares-da Rosa, S. (2017). Single-Domain Antibodies As Versatile Affinity Reagents for Analytical and Diagnostic Applications. Front. Immunol., 8.","DOI":"10.3389\/fimmu.2017.00977"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1038\/363446a0","article-title":"Naturally occurring antibodies devoid of light chains","volume":"363","author":"Atarhouch","year":"1993","journal-title":"Nature"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"775","DOI":"10.1146\/annurev-biochem-063011-092449","article-title":"Nanobodies: Natural Single-Domain Antibodies","volume":"82","author":"Muyldermans","year":"2013","journal-title":"Annu. Rev. Biochem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"5985","DOI":"10.1007\/s00216-016-9585-x","article-title":"VHH antibodies: Emerging reagents for the analysis of environmental chemicals","volume":"408","author":"Bever","year":"2016","journal-title":"Anal. Bioanal. Chem."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"132701","DOI":"10.1016\/j.jhazmat.2023.132701","article-title":"Protein nanoscaffold enables programmable nanobody-luciferase immunoassembly for sensitive and simultaneous detection of aflatoxin B1 and ochratoxin A","volume":"462","author":"Wu","year":"2024","journal-title":"J. Hazard. Mater."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"116760","DOI":"10.1016\/j.microc.2025.116760","article-title":"Nanobody-based nanoarchitectonics of ultra-sensitive indirect competitive chemiluminescent enzyme immunoassay for aflatoxin B1 in cereals","volume":"220","author":"Ke","year":"2026","journal-title":"Microchem. J."},{"key":"ref_11","first-page":"e2755","article-title":"Structure and specificity of several triclocarban-binding single domain camelid antibody fragments","volume":"32","author":"Wogulis","year":"2018","journal-title":"J. Mol. Recognit."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"38","DOI":"10.1016\/j.jgg.2022.04.010","article-title":"Characterization of heavy-chain antibody gene repertoires in Bactrian camels","volume":"50","author":"Liu","year":"2023","journal-title":"J. Genet. Genom."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"11306","DOI":"10.1021\/acs.analchem.3c01370","article-title":"Structural Insights into the Stability and Recognition Mechanism of the Antiquinalphos Nanobody for the Detection of Quinalphos in Foods","volume":"95","author":"Li","year":"2023","journal-title":"Anal. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"188","DOI":"10.1016\/j.ijbiomac.2021.11.182","article-title":"Enhancing the detection sensitivity of nanobody against aflatoxin B1 through structure-guided modification","volume":"194","author":"He","year":"2022","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_15","first-page":"349","article-title":"Development of the Detection Method for Aflatoxin B1 Based on Nanobody-Nano Luciferase Fusion Proteins","volume":"24","author":"Chen","year":"2024","journal-title":"J. Chin. Inst. Food Sci. Technol."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"141220","DOI":"10.1016\/j.foodchem.2024.141220","article-title":"Development of a bioluminescent immunoassay based on Fc-specific conjugated antibody\u2013nanoluciferase immunoreagents for determining aflatoxin B1","volume":"463","author":"Du","year":"2025","journal-title":"Food Chem."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2363","DOI":"10.1039\/D1SC06825C","article-title":"The importance of the compact disordered state in the fuzzy interactions between intrinsically disordered proteins","volume":"13","author":"Wang","year":"2022","journal-title":"Chem. Sci."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"235","DOI":"10.1093\/nar\/28.1.235","article-title":"The Protein Data Bank","volume":"28","author":"Berman","year":"2000","journal-title":"Nucleic Acids Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1007\/s12104-018-9855-y","article-title":"Chemical shift assignments of a camelid nanobody against aflatoxin B1","volume":"13","author":"Nie","year":"2018","journal-title":"Biomol. NMR Assign."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"W296","DOI":"10.1093\/nar\/gky427","article-title":"SWISS-MODEL: Homology modelling of protein structures and complexes","volume":"46","author":"Waterhouse","year":"2018","journal-title":"Nucleic Acids Res."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"D1373","DOI":"10.1093\/nar\/gkac956","article-title":"PubChem 2023 update","volume":"51","author":"Kim","year":"2023","journal-title":"Nucleic Acids Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3219","DOI":"10.1038\/s41596-024-01011-0","article-title":"The HADDOCK2.4 web server for integrative modeling of biomolecular complexes","volume":"19","author":"Honorato","year":"2024","journal-title":"Nat. Protoc."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1016\/j.softx.2015.06.001","article-title":"GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers","volume":"1\u20132","author":"Abraham","year":"2015","journal-title":"SoftwareX"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"2135","DOI":"10.1002\/jcc.23354","article-title":"CHARMM36 all-atom additive protein force field: Validation based on comparison to NMR data","volume":"34","author":"Huang","year":"2013","journal-title":"J. Comput. Chem."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"4089","DOI":"10.1021\/acs.jcim.4c00126","article-title":"Exploring the Limits of the Generalized CHARMM and AMBER Force Fields through Predictions of Hydration Free Energy of Small Molecules","volume":"64","author":"Chakravorty","year":"2024","journal-title":"J. Chem. Inf. Model."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"926","DOI":"10.1063\/1.445869","article-title":"Comparison of simple potential functions for simulating liquid water","volume":"79","author":"Jorgensen","year":"1983","journal-title":"J. Chem. Phys."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"014101","DOI":"10.1063\/1.2408420","article-title":"Canonical sampling through velocity rescaling","volume":"126","author":"Bussi","year":"2007","journal-title":"J. Chem. Phys."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"116202","DOI":"10.7498\/aps.69.20200323","article-title":"Molecular dynamics simulation of shock-induced isostructural phase transition in single crystal Ce","volume":"69","author":"Diwu","year":"2020","journal-title":"Acta Phys. Sin."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1592","DOI":"10.1021\/acs.jctc.2c01032","article-title":"Optimal Bond Constraint Topology for Molecular Dynamics Simulations of Cholesterol","volume":"19","author":"Thallmair","year":"2023","journal-title":"J. Chem. Theory Comput."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"244107","DOI":"10.1063\/5.0095581","article-title":"Flexible multipole moments in smooth particle mesh Ewald","volume":"156","author":"Symons","year":"2022","journal-title":"J. Chem. Phys."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1186\/s13321-024-00918-w","article-title":"StreaMD: The toolkit for high-throughput molecular dynamics simulations","volume":"16","author":"Ivanova","year":"2024","journal-title":"J. Cheminform."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"125102","DOI":"10.1063\/5.0019088","article-title":"A workflow for exploring ligand dissociation from a macromolecule: Efficient random acceleration molecular dynamics simulation and interaction fingerprint analysis of ligand trajectories","volume":"153","author":"Kokh","year":"2020","journal-title":"J. Chem. Phys."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"6281","DOI":"10.1021\/acs.jctc.1c00645","article-title":"gmx_MMPBSA: A New Tool to Perform End-State Free Energy Calculations with GROMACS","volume":"17","author":"Valiente","year":"2021","journal-title":"J. Chem. Theory Comput."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"390","DOI":"10.1007\/s00894-024-06189-4","article-title":"A head-to-head comparison of MM\/PBSA and MM\/GBSA in predicting binding affinities for the CB1 cannabinoid ligands","volume":"30","author":"Yau","year":"2024","journal-title":"J. Mol. Model."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"7902","DOI":"10.1021\/acs.jafc.5c10020","article-title":"Conformational Dynamics-Driven Engineering of Nanobody Biosensor for Enhanced Detection of Ochratoxin A","volume":"74","author":"Wu","year":"2026","journal-title":"J. Agric. Food Chem."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1186\/s13036-023-00350-y","article-title":"Development of anti-aflatoxin B1 nanobodies from a novel mutagenesis-derived synthetic library for traditional Chinese medicine and foods safety testing","volume":"17","author":"Lee","year":"2023","journal-title":"J. Biol. Eng."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/9\/1265\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T04:13:21Z","timestamp":1789013601000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/9\/1265"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,1]]},"references-count":36,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["biom16091265"],"URL":"https:\/\/doi.org\/10.3390\/biom16091265","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,1]]}}}