{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,24]],"date-time":"2026-10-24T01:46:12Z","timestamp":1792806372998,"version":"4.5.0"},"reference-count":48,"publisher":"MDPI AG","issue":"23","license":[{"start":{"date-parts":[[2020,11,24]],"date-time":"2020-11-24T00:00:00Z","timestamp":1606176000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Vilnius University","award":["MSF-JM-3"],"award-info":[{"award-number":["MSF-JM-3"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>The formation of amyloid fibrils is linked to multiple neurodegenerative disorders, including Alzheimer\u2019s and Parkinson\u2019s disease. Despite years of research and countless studies on the topic of such aggregate formation, as well as their resulting structure, the current knowledge is still fairly limited. One of the main aspects prohibiting effective aggregation tracking is the environment\u2019s effect on amyloid-specific dyes, namely thioflavin-T (ThT). Currently, there are only a few studies hinting at ionic strength being one of the factors that modulate the dye\u2019s binding affinity and fluorescence intensity. In this work we explore this effect under a range of ionic strength conditions, using insulin, lysozyme, mouse prion protein, and \u03b1-synuclein fibrils. We show that ionic strength is an extremely important factor affecting both the binding affinity, as well as the fluorescence intensity of ThT.<\/jats:p>","DOI":"10.3390\/ijms21238916","type":"journal-article","created":{"date-parts":[[2020,11,24]],"date-time":"2020-11-24T20:50:22Z","timestamp":1606251022000},"page":"8916","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":59,"title":["Effect of Ionic Strength on Thioflavin-T Affinity to Amyloid Fibrils and Its Fluorescence Intensity"],"prefix":"10.3390","volume":"21","author":[{"given":"Kamile","family":"Mikalauskaite","sequence":"first","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University, LT-10257 Vilnius, Lithuania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mantas","family":"Ziaunys","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University, LT-10257 Vilnius, Lithuania"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5285-871X","authenticated-orcid":false,"given":"Tomas","family":"Sneideris","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University, LT-10257 Vilnius, Lithuania"},{"name":"Department of Chemistry, University of Cambridge, Cambridge CB2 1EW, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1829-5455","authenticated-orcid":false,"given":"Vytautas","family":"Smirnovas","sequence":"additional","affiliation":[{"name":"Institute of Biotechnology, Life Sciences Center, Vilnius University, LT-10257 Vilnius, Lithuania"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,11,24]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1038\/nrm3810","article-title":"The amyloid state and its association with protein misfolding diseases","volume":"15","author":"Knowles","year":"2014","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"27","DOI":"10.1146\/annurev-biochem-061516-045115","article-title":"Protein Misfolding, Amyloid Formation, and Human Disease: A Summary of Progress Over the Last Decade","volume":"86","author":"Chiti","year":"2017","journal-title":"Annu. Rev. Biochem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/B978-0-12-386483-3.00003-3","article-title":"Lysozyme: A model protein for amyloid research","volume":"84","author":"Swaminathan","year":"2011","journal-title":"Adv. Protein Chem. Struct. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"552","DOI":"10.1016\/j.ijbiomac.2018.06.109","article-title":"Effect of fibrillation conditions on the anti-amyloidogenic properties of polyphenols and their involved mechanisms","volume":"118","author":"Mahdavimehr","year":"2018","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1126\/science.1105850","article-title":"Self-propagating, molecular-level polymorphism in Alzheimer\u2019s beta-amyloid fibrils","volume":"307","author":"Petkova","year":"2005","journal-title":"Science"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"883","DOI":"10.1111\/j.1471-4159.2012.07847.x","article-title":"Cross-seeding effects of amyloid \u03b2-protein and \u03b1-synuclein","volume":"122","author":"Ono","year":"2012","journal-title":"J. Neurochem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"5950","DOI":"10.1111\/j.1742-4658.2005.05025.x","article-title":"Structures for amyloid fibrils","volume":"272","author":"Makin","year":"2005","journal-title":"FEBS J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"272","DOI":"10.1016\/j.trci.2019.05.008","article-title":"Alzheimer\u2019s disease drug development pipeline: 2019","volume":"5","author":"Cummings","year":"2019","journal-title":"Alzheimer\u2019s Dement Transl. Res. Clin. Interv."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"735","DOI":"10.1080\/13543784.2017.1323868","article-title":"Why do trials for Alzheimer\u2019s disease drugs keep failing? A discontinued drug perspective for 2010\u20132015","volume":"26","author":"Mehta","year":"2017","journal-title":"Expert Opin. Investig. Drugs"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"7615","DOI":"10.1021\/bi100745j","article-title":"Salt-Induced Modulation of the Pathway of Amyloid Fibril Formation by the Mouse Prion Protein","volume":"49","author":"Jain","year":"2010","journal-title":"Biochemistry"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Iannuzzi, C., Borriello, M., Portaccio, M., Irace, G., and Sirangelo, I. (2017). Insights into insulin fibril assembly at physiological and acidic ph and related amyloid intrinsic fluorescence. Int. J. Mol. Sci., 18.","DOI":"10.3390\/ijms18122551"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"18349","DOI":"10.1073\/pnas.0806270105","article-title":"Molecular structural basis for polymorphism in Alzheimer\u2019s -amyloid fibrils","volume":"105","author":"Paravastu","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1016\/j.abb.2019.02.001","article-title":"Atomic force microscopy for single molecule characterisation of protein aggregation","volume":"664","author":"Ruggeri","year":"2019","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"121","DOI":"10.1007\/978-1-4939-7811-3_6","article-title":"An Aggregate Weight-Normalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly Intermediates","volume":"1777","author":"Wetzel","year":"2018","journal-title":"Methods Mol. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1080\/13506129.2017.1304905","article-title":"ThT 101: A primer on the use of thioflavin T to investigate amyloid formation","volume":"24","author":"Weber","year":"2017","journal-title":"Amyloid"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"16490","DOI":"10.1021\/la303677t","article-title":"Conformational analysis of thioflavin T bound to the surface of amyloid fibrils","volume":"28","author":"Robbins","year":"2012","journal-title":"Langmuir"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1405","DOI":"10.1016\/j.bbapap.2010.04.001","article-title":"Molecular mechanism of Thioflavin-T binding to amyloid fibrils","volume":"1804","author":"Biancalana","year":"2010","journal-title":"Biochim. Biophys. Acta Proteins Proteom."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Manno, M., Craparo, E.F., Martorana, V., Bulone, D., and San Biagio, P.L. (2006). Kinetics of Insulin Aggregation: Disentanglement of Amyloid Fibrillation from Large-Size Cluster Formation. Biophys. J.","DOI":"10.1529\/biophysj.105.077636"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"382","DOI":"10.1002\/pro.3549","article-title":"Suppression, disaggregation, and modulation of \u03b3-Synuclein fibrillation pathway by green tea polyphenol EGCG","volume":"28","author":"Roy","year":"2019","journal-title":"Protein Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"8475","DOI":"10.1021\/am501341u","article-title":"Epigallocatechin-3-gallate (EGCG)-Stabilized Selenium Nanoparticles Coated with Tet-1 Peptide To Reduce Amyloid-\u03b2 Aggregation and Cytotoxicity","volume":"6","author":"Zhang","year":"2014","journal-title":"ACS Appl. Mater. Interfaces"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"5960","DOI":"10.1111\/j.1742-4658.2009.07307.x","article-title":"The thioflavin T fluorescence assay for amyloid fibril detection can be biased by the presence of exogenous compounds","volume":"276","author":"Hudson","year":"2009","journal-title":"FEBS J."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3492","DOI":"10.1016\/j.bpj.2010.08.074","article-title":"Interactions between Amyloidophilic Dyes and Their Relevance to Studies of Amyloid Inhibitors","volume":"99","author":"Buell","year":"2010","journal-title":"Biophys. J."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"15174","DOI":"10.1021\/jp805560c","article-title":"Thioflavin T Hydroxylation at Basic pH and Its Effect on Amyloid Fibril Detection","volume":"112","author":"Groenning","year":"2008","journal-title":"J. Phys. Chem. B"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"4572","DOI":"10.1038\/s41598-020-61663-2","article-title":"Formation of distinct prion protein amyloid fibrils under identical experimental conditions","volume":"10","author":"Ziaunys","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"e8208","DOI":"10.7717\/peerj.8208","article-title":"Concentration-dependent polymorphism of insulin amyloid fibrils","volume":"7","author":"Sakalauskas","year":"2019","journal-title":"PeerJ"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1080\/13506129.2018.1517736","article-title":"Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in \u03b1-synuclein fibrils","volume":"25","author":"Sidhu","year":"2018","journal-title":"Amyloid"},{"key":"ref_27","doi-asserted-by":"crossref","unstructured":"Ziaunys, M., Mikalauskaite, K., and Smirnovas, V. (2019). Amyloidophilic Molecule Interactions on the Surface of Insulin Fibrils: Cooperative Binding and Fluorescence Quenching. Sci. Rep.","DOI":"10.1038\/s41598-019-56788-y"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Sulatskaya, A.I., Kuznetsova, I.M., Belousov, M.V., Bondarev, S.A., Zhouravleva, G.A., and Turoverov, K.K. (2016). Stoichiometry and Affinity of Thioflavin T Binding to Sup35p Amyloid Fibrils. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0156314"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"483","DOI":"10.1016\/j.jsb.2007.06.004","article-title":"Binding mode of Thioflavin T in insulin amyloid fibrils","volume":"159","author":"Groenning","year":"2007","journal-title":"J. Struct. Biol."},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"Kuznetsova, I.M., Sulatskaya, A.I., Uversky, V.N., and Turoverov, K.K. (2012). Analyzing Thioflavin T Binding to Amyloid Fibrils by an Equilibrium Microdialysis-Based Technique. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0030724"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/srep14651","article-title":"A covalent homodimer probing early oligomers along amyloid aggregation","volume":"5","author":"Halabelian","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/j.ijbiomac.2018.09.165","article-title":"Influence of the ionic strength on the amyloid fibrillogenesis of hen egg white lysozyme","volume":"121","author":"Wawer","year":"2019","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1016\/j.jsb.2008.02.002","article-title":"On the binding of Thioflavin-T to HET-s amyloid fibrils assembled at pH 2","volume":"162","author":"Lascu","year":"2008","journal-title":"J. Struct. Biol."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"306","DOI":"10.1006\/abbi.1997.0137","article-title":"Stopped-flow kinetics reveal multiple phases of thioflavin T binding to Alzheimer \u03b2(1-40) amyloid fibrils. Arch","volume":"342","author":"LeVine","year":"1997","journal-title":"Biochem. Biophys."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"019008","DOI":"10.1116\/1.4941008","article-title":"Dissecting ion-specific from electrostatic salt effects on amyloid fibrillation: A case study of insulin","volume":"11","author":"Kutsch","year":"2016","journal-title":"Biointerphases"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1362","DOI":"10.1021\/bm0498979","article-title":"Guanidine Hydrochloride Can Induce Amyloid Fibril Formation from Hen Egg-White Lysozyme","volume":"5","author":"Vernaglia","year":"2004","journal-title":"Biomacromolecules"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"11404","DOI":"10.1021\/bi034868o","article-title":"Partially Folded Intermediates in Insulin Fibrillation","volume":"42","author":"Ahmad","year":"2003","journal-title":"Biochemistry"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Milto, K., Michailova, K., and Smirnovas, V. (2014). Elongation of Mouse Prion Protein Amyloid-Like Fibrils: Effect of Temperature and Denaturant Concentration. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0094469"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"517","DOI":"10.1021\/js960297s","article-title":"Toward Understanding Insulin Fibrillation","volume":"86","author":"Brange","year":"1997","journal-title":"J. Pharm. Sci."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1111\/j.1365-2796.2011.02387.x","article-title":"Prions and protein-folding diseases","volume":"270","author":"Norrby","year":"2011","journal-title":"J. Intern. Med."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"a009399","DOI":"10.1101\/cshperspect.a009399","article-title":"\u03b1-Synuclein in Parkinson\u2019s Disease","volume":"2","author":"Stefanis","year":"2012","journal-title":"Cold Spring Harb. Perspect. Med."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"2170","DOI":"10.1021\/acs.biochem.7b00035","article-title":"Binding of Thioflavin-T to Amyloid Fibrils Leads to Fluorescence Self-Quenching and Fibril Compaction","volume":"56","author":"Lindberg","year":"2017","journal-title":"Biochemistry"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1073","DOI":"10.1016\/j.bbabio.2007.06.004","article-title":"Infrared spectroscopy of proteins","volume":"1767","author":"Barth","year":"2007","journal-title":"Biochim. Biophys. Acta Bioenergy"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Lee, G., Lee, W., Lee, H., Woo Lee, S., Sung Yoon, D., Eom, K., and Kwon, T. (2012). Mapping the surface charge distribution of amyloid fibril. Appl. Phys. Lett., 101.","DOI":"10.1063\/1.4739494"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Sneideris, T., Sakalauskas, A., Sternke-Hoffmann, R., Peduzzo, A., Ziaunys, M., Buell, A.K., and Smirnovas, V. (2019). The Environment Is a Key Factor in Determining the Anti-Amyloid Efficacy of EGCG. Biomolecules, 9.","DOI":"10.3390\/biom9120855"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"e1271","DOI":"10.7717\/peerj.1271","article-title":"Looking for a generic inhibitor of amyloid-like fibril formation among flavone derivatives","volume":"3","author":"Cannon","year":"2015","journal-title":"PeerJ"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"11119","DOI":"10.1073\/pnas.0901422106","article-title":"Ultrasonication-dependent production and breakdown lead to minimum-sized amyloid fibrils","volume":"106","author":"Chatani","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"8727","DOI":"10.1021\/acs.jpcb.9b08652","article-title":"Additional Thioflavin-T Binding Mode in Insulin Fibril Inner Core Region","volume":"123","author":"Ziaunys","year":"2019","journal-title":"J. Phys. Chem. 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