{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T06:41:09Z","timestamp":1789713669476,"version":"4.0.1"},"reference-count":51,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2017,8,12]],"date-time":"2017-08-12T00:00:00Z","timestamp":1502496000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Toxins"],"abstract":"<jats:p>Spider gating modifier toxins (GMTs) are potent modulators of voltage-gated ion channels and have thus attracted attention as drug leads for several pathophysiological conditions. GMTs contain three disulfide bonds organized in an inhibitory cystine knot, which putatively confers them with high stability; however, thus far, there has not been a focused study to establish the stability of GMTs in physiological conditions. We examined the resistance of five GMTs including GpTx-1, HnTx-IV, HwTx-IV, PaurTx-3 and SgTx-1, to pH, thermal and proteolytic degradation. The peptides were stable under physiological conditions, except SgTx-1, which was susceptible to proteolysis, probably due to a longer C-terminus compared to the other peptides. In non-physiological conditions, the five peptides withstood chaotropic degradation, and all but SgTx-1 remained intact after prolonged exposure to high temperature; however, the peptides were degraded in strongly alkaline solutions. GpTx-1 and PaurTx-3 were more resistant to basic hydrolysis than HnTx-IV, HwTx-IV and SgTx-1, probably because a shorter interconnecting loop 3 on GpTx-1 and PaurTx-3 may stabilize interactions between the C-terminus and the hydrophobic patch. Here, we establish that most GMTs are exceptionally stable, and propose that, in the design of GMT-based therapeutics, stability can be enhanced by optimizing the C-terminus in terms of length, and increased interactions with the hydrophobic patch.<\/jats:p>","DOI":"10.3390\/toxins9080248","type":"journal-article","created":{"date-parts":[[2017,8,14]],"date-time":"2017-08-14T10:23:12Z","timestamp":1502706192000},"page":"248","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":24,"title":["Lengths of the C-Terminus and Interconnecting Loops Impact Stability of Spider-Derived Gating Modifier Toxins"],"prefix":"10.3390","volume":"9","author":[{"given":"Akello","family":"Agwa","sequence":"first","affiliation":[{"name":"Institute for Molecular Bioscience, the University of Queensland, Brisbane, Queensland 4072, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yen-Hua","family":"Huang","sequence":"additional","affiliation":[{"name":"Institute for Molecular Bioscience, the University of Queensland, Brisbane, Queensland 4072, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Craik","sequence":"additional","affiliation":[{"name":"Institute for Molecular Bioscience, the University of Queensland, Brisbane, Queensland 4072, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9564-9651","authenticated-orcid":false,"given":"S\u00f3nia","family":"Henriques","sequence":"additional","affiliation":[{"name":"Institute for Molecular Bioscience, the University of Queensland, Brisbane, Queensland 4072, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christina","family":"Schroeder","sequence":"additional","affiliation":[{"name":"Institute for Molecular Bioscience, the University of Queensland, Brisbane, Queensland 4072, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2017,8,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"King, G.F. (2015). Therapeutic applications of spider-venom peptides. Venoms to Drugs: Venom as a Source for the Development of Human Therapeutics, Royal Society of Chemistry.","DOI":"10.1039\/9781849737876"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"823","DOI":"10.1517\/17460441.2.6.823","article-title":"Spider peptide toxins as leads for drug development","volume":"2","author":"Escoubas","year":"2007","journal-title":"Expert Opin. Drug Discov."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"124","DOI":"10.1016\/j.toxicon.2006.09.022","article-title":"Voltage-gated ion channels and gating modifier toxins","volume":"49","author":"Catterall","year":"2007","journal-title":"Toxicon"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"73","DOI":"10.1016\/j.pharmthera.2016.11.015","article-title":"NaV1.7 as a pain target\u2014From gene to pharmacology","volume":"172","author":"Vetter","year":"2017","journal-title":"Pharmacol. Ther."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"494","DOI":"10.1038\/nature17976","article-title":"Selective spider toxins reveal a role for the NaV1.1 channel in mechanical pain","volume":"534","author":"Osteen","year":"2016","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Cox, B., and Gosling, M. (2015). Does nature do ion channel drug discovery better than us?. Ion Channel Drug Discovery, Royal Society of Chemistry.","DOI":"10.1039\/9781849735087"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"2577","DOI":"10.1113\/jphysiol.2011.224204","article-title":"Voltage-gated sodium channels at 60: Structure, function and pathophysiology","volume":"590","author":"Catterall","year":"2012","journal-title":"J. Physiol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1146\/annurev-neuro-060909-153234","article-title":"Sodium channels in normal and pathological pain","volume":"33","author":"Cummins","year":"2010","journal-title":"Annu. Rev. Neurosci."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"39662","DOI":"10.1038\/srep39662","article-title":"Insensitivity to pain induced by a potent selective closed-state NaV1.7 inhibitor","volume":"7","author":"Flinspach","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1016\/j.cbpa.2017.03.004","article-title":"Progress and challenges in the optimization of toxin peptides for development as pain therapeutics","volume":"38","author":"Netirojjanakul","year":"2017","journal-title":"Curr. Opin. Chem. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"6836","DOI":"10.1073\/pnas.1621263114","article-title":"Pharmacology of the Nav1.1 domain IV voltage sensor reveals coupling between inactivation gating processes","volume":"114","author":"Osteen","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Agwa, A.J., Henriques, S.T., and Schroeder, C.I. (2017). Gating modifier toxin interactions with ion channels and lipid bilayers: is the trimolecular complex real?. Neuropharmacology, in press.","DOI":"10.1016\/j.neuropharm.2017.04.004"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"361","DOI":"10.1081\/TXR-120014409","article-title":"Structure and function of insecticidal neurotoxins from Australian funnel-web spiders","volume":"21","author":"King","year":"2002","journal-title":"J. Toxicol. Toxin Rev."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"43","DOI":"10.1016\/S0041-0101(00)00160-4","article-title":"The cystine knot motif in toxins and implications for drug design","volume":"39","author":"Craik","year":"2001","journal-title":"Toxicon"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1573","DOI":"10.1016\/S0041-0101(98)00149-4","article-title":"The cystine knot structure of ion channel toxins and related polypeptides","volume":"36","author":"Norton","year":"1998","journal-title":"Toxicon"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1833","DOI":"10.1002\/pro.5560031022","article-title":"A common structural motif incorporating a cystine knot and a triple-stranded beta-sheet in toxic and inhibitory polypeptides","volume":"3","author":"Pallaghy","year":"1994","journal-title":"Protein Sci."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"2299","DOI":"10.1021\/jm501765v","article-title":"Engineering potent and selective analogues of GpTx-1, a tarantula venom peptide antagonist of the NaV1.7 sodium channel","volume":"58","author":"Murray","year":"2015","journal-title":"J. Med. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"890","DOI":"10.1021\/bi0353373","article-title":"Solution structure and functional characterization of SGTx1, a modifier of KV2.1 channel gating","volume":"43","author":"Lee","year":"2004","journal-title":"Biochemistry"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"37734","DOI":"10.1074\/jbc.M405765200","article-title":"Structure-activity relationships of hainantoxin-IV and structure determination of active and inactive sodium channel blockers","volume":"279","author":"Li","year":"2004","journal-title":"J. Biol. Chem."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"17049","DOI":"10.1074\/jbc.M116.729095","article-title":"Interaction of tarantula venom peptide ProTx-II with lipid membranes is a prerequisite for its inhibition of human voltage-gated sodium channel NaV1.7","volume":"29","author":"Henriques","year":"2016","journal-title":"J. Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"22707","DOI":"10.1074\/jbc.M113.461392","article-title":"Analysis of the structural and molecular basis of voltage-sensitive sodium channel inhibition by the spider toxin huwentoxin-IV (mu-TRTX-Hh2a)","volume":"288","author":"Minassian","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"40883","DOI":"10.1038\/srep40883","article-title":"Pharmacological characterisation of the highly NaV1.7 selective spider venom peptide Pn3a","volume":"7","author":"Deuis","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2445","DOI":"10.1111\/bph.13081","article-title":"Seven novel modulators of the analgesic target NaV1.7 uncovered using a high-throughput venom-based discovery approach","volume":"172","author":"Klint","year":"2015","journal-title":"Br. J. Pharmacol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"419","DOI":"10.1124\/mol.105.015941","article-title":"Four novel tarantula toxins as selective modulators of voltage-gated sodium channel subtypes","volume":"69","author":"Bosmans","year":"2006","journal-title":"Mol. Pharmacol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1190","DOI":"10.1046\/j.0014-2956.2002.02760.x","article-title":"The solution structure of gomesin, an antimicrobial cysteine-rich peptide from the spider","volume":"269","author":"Mandard","year":"2002","journal-title":"Eur. J. Biochem."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"835","DOI":"10.1016\/j.bbamem.2017.01.020","article-title":"Spider peptide toxin HwTx-IV engineered to bind to lipid membranes has an increased inhibitory potency at human voltage-gated sodium channel hNaV1.7","volume":"1859","author":"Agwa","year":"2017","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"872","DOI":"10.1016\/j.bbamem.2016.02.002","article-title":"Membrane-binding properties of gating modifier and pore-blocking toxins: Membrane interaction is not a prerequisite for modification of channel gating","volume":"1858","author":"Deplazes","year":"2016","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"34333","DOI":"10.1038\/srep34333","article-title":"Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels","volume":"6","author":"Lau","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"857","DOI":"10.1038\/nature03873","article-title":"Voltage-sensor activation with a tarantula toxin as cargo","volume":"436","author":"Milescu","year":"2005","journal-title":"Nature"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"232","DOI":"10.1038\/nature02632","article-title":"A membrane-access mechanism of ion channel inhibition by voltage sensor toxins from spider venom","volume":"430","author":"Lee","year":"2004","journal-title":"Nature"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"23894","DOI":"10.1038\/srep23894","article-title":"Molecular interactions between tarantula toxins and low-voltage-activated calcium channels","volume":"6","author":"Salari","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"4366","DOI":"10.3390\/toxins7104366","article-title":"The cystine knot is responsible for the exceptional stability of the insecticidal spider toxin omega-hexatoxin-Hv1a","volume":"7","author":"Herzig","year":"2015","journal-title":"Toxins"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"202","DOI":"10.1038\/nature07473","article-title":"Deconstructing voltage sensor function and pharmacology in sodium channels","volume":"456","author":"Bosmans","year":"2008","journal-title":"Nature"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"175","DOI":"10.1016\/j.tips.2009.12.007","article-title":"Targeting sodium channel voltage sensors with spider toxins","volume":"31","author":"Bosmans","year":"2010","journal-title":"Trends Pharmacol. Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"W375","DOI":"10.1093\/nar\/gkm216","article-title":"MolProbity: all-atom contacts and structure validation for proteins and nucleic acids","volume":"35","author":"Davis","year":"2007","journal-title":"Nucleic Acids Res."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"29","DOI":"10.1016\/0263-7855(96)00009-4","article-title":"MOLMOL: A program for display and analysis of macromolecular structures","volume":"14","author":"Koradi","year":"1996","journal-title":"J. Mol. Graph."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"944","DOI":"10.1016\/j.bbamem.2012.12.002","article-title":"Design and characterization of novel antimicrobial peptides, R-BP100 and RW-BP100, with activity against Gram-negative and Gram-positive bacteria","volume":"1828","author":"Torcato","year":"2013","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"5965","DOI":"10.1021\/bi049711q","article-title":"Thermal, chemical, and enzymatic stability of the cyclotide kalata B1: The importance of the cyclic cystine knot","volume":"43","author":"Colgrave","year":"2004","journal-title":"Biochemistry"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Er, S.Y., Cristofori-Armstrong, B., Escoubas, P., and Rash, L.D. (2017). Discovery and molecular interaction studies of a highly stable, tarantula peptide modulator of acid-sensing ion channel 1. Neuropharmacology.","DOI":"10.1016\/j.neuropharm.2017.03.020"},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Hardy, M.C., Daly, N.L., Mobli, M., Morales, R.A.V., and King, G.F. (2013). Isolation of an orally active insecticidal toxin from the venom of an australian tarantula. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0073136"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"7292","DOI":"10.1021\/bi960341i","article-title":"Guanidine hydrochloride unfolding of peptide helices: separation of denaturant and salt effects","volume":"35","author":"Smith","year":"1996","journal-title":"Biochemistry"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"16689","DOI":"10.1021\/ja903478s","article-title":"Preferential interactions of guanidinum ions with aromatic groups over aliphatic groups","volume":"131","author":"Mason","year":"2009","journal-title":"J. Am. Chem. Soc."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"98","DOI":"10.1016\/S0167-4838(02)00341-2","article-title":"The denaturation of circular enterocin AS-48 by urea and guanidinium hydrochloride","volume":"1598","author":"Cobos","year":"2002","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"643","DOI":"10.1002\/psc.2451","article-title":"A positively charged surface patch is important for hainantoxin-IV binding to voltage-gated sodium channels","volume":"18","author":"Liu","year":"2012","journal-title":"J. Pept. Sci."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"27300","DOI":"10.1074\/jbc.M708447200","article-title":"Tarantula huwentoxin-IV inhibits neuronal sodium channels by binding to receptor site 4 and trapping the domain II voltage sensor in the closed configuration","volume":"283","author":"Xiao","year":"2008","journal-title":"J. Biol. Chem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"687","DOI":"10.1002\/prot.20449","article-title":"The CCPN data model for NMR spectroscopy: development of a software pipeline","volume":"59","author":"Vranken","year":"2005","journal-title":"Proteins"},{"key":"ref_47","doi-asserted-by":"crossref","unstructured":"W\u00fcthrich, K. (1986). NMR of Proteins and Nucleic Acids, Wiley Interscience.","DOI":"10.1051\/epn\/19861701011"},{"key":"ref_48","first-page":"353","article-title":"Automated NMR structure calculation with CYANA","volume":"278","year":"2004","journal-title":"Methods Mol. Biol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1007\/s10858-013-9741-y","article-title":"Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks","volume":"56","author":"Shen","year":"2013","journal-title":"J. Biomol. NMR"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"662","DOI":"10.1002\/prot.20408","article-title":"RECOORD: A recalculated coordinate database of 500+ proteins from the PDB using restraints from the BioMagResBank","volume":"59","author":"Nederveen","year":"2005","journal-title":"Proteins"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"905","DOI":"10.1107\/S0907444998003254","article-title":"Crystallography & NMR system: A new software suite for macromolecular structure determination","volume":"54","author":"Adams","year":"1998","journal-title":"Acta Crystall. D Biol. Crystall."}],"container-title":["Toxins"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2072-6651\/9\/8\/248\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T18:42:12Z","timestamp":1760208132000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2072-6651\/9\/8\/248"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2017,8,12]]},"references-count":51,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2017,8]]}},"alternative-id":["toxins9080248"],"URL":"https:\/\/doi.org\/10.3390\/toxins9080248","relation":{},"ISSN":["2072-6651"],"issn-type":[{"value":"2072-6651","type":"electronic"}],"subject":[],"published":{"date-parts":[[2017,8,12]]}}}