{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,2]],"date-time":"2026-03-02T16:54:19Z","timestamp":1772470459005,"version":"3.50.1"},"reference-count":44,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2021,5,27]],"date-time":"2021-05-27T00:00:00Z","timestamp":1622073600000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2021,5,27]],"date-time":"2021-05-27T00:00:00Z","timestamp":1622073600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"name":"Polish National Science Center","award":["grant No. N N301 044939"],"award-info":[{"award-number":["grant No. N N301 044939"]}]},{"name":"Polish National Science Center","award":["grant No. N N301 044939"],"award-info":[{"award-number":["grant No. N N301 044939"]}]},{"name":"Polish National Science Center","award":["grant No. N N301 044939"],"award-info":[{"award-number":["grant No. N N301 044939"]}]},{"name":"Polish National Science Center","award":["grant No. N N301 044939"],"award-info":[{"award-number":["grant No. N N301 044939"]}]},{"name":"Polish National Science Center","award":["grant No. N N301 044939"],"award-info":[{"award-number":["grant No. N N301 044939"]}]},{"name":"Polish National Science Center","award":["grant No. N N301 044939"],"award-info":[{"award-number":["grant No. N N301 044939"]}]},{"name":"Polish Ministry of Science and Higher Education","award":["163500\/BF, BST 173300\/BF task 34, BST 0180100 03\/BF task 22, 501 D111 01 1110105 (PP\/BF)"],"award-info":[{"award-number":["163500\/BF, BST 173300\/BF task 34, BST 0180100 03\/BF task 22, 501 D111 01 1110105 (PP\/BF)"]}]},{"name":"Polish Ministry of Science and Higher Education","award":["163500\/BF, BST 173300\/BF task 34, BST 0180100 03\/BF task 22, 501 D111 01 1110105 (PP\/BF)"],"award-info":[{"award-number":["163500\/BF, BST 173300\/BF task 34, BST 0180100 03\/BF task 22, 501 D111 01 1110105 (PP\/BF)"]}]},{"name":"Polish Ministry of Science and Higher Education","award":["163500\/BF, BST 173300\/BF task 34, BST 0180100 03\/BF task 22, 501 D111 01 1110105 (PP\/BF)"],"award-info":[{"award-number":["163500\/BF, BST 173300\/BF task 34, BST 0180100 03\/BF task 22, 501 D111 01 1110105 (PP\/BF)"]}]},{"name":"Polish Ministry of Science and Higher Education","award":["163500\/BF, BST 173300\/BF task 34, BST 0180100 03\/BF task 22, 501 D111 01 1110105 (PP\/BF)"],"award-info":[{"award-number":["163500\/BF, BST 173300\/BF task 34, BST 0180100 03\/BF task 22, 501 D111 01 1110105 (PP\/BF)"]}]},{"name":"Polish Ministry of Science and Higher Education","award":["163500\/BF, BST 173300\/BF task 34, BST 0180100 03\/BF task 22, 501 D111 01 1110105 (PP\/BF)"],"award-info":[{"award-number":["163500\/BF, BST 173300\/BF task 34, BST 0180100 03\/BF task 22, 501 D111 01 1110105 (PP\/BF)"]}]},{"name":"Polish Ministry of Science and Higher Education","award":["163500\/BF, BST 173300\/BF task 34, BST 0180100 03\/BF task 22, 501 D111 01 1110105 (PP\/BF)"],"award-info":[{"award-number":["163500\/BF, BST 173300\/BF task 34, BST 0180100 03\/BF task 22, 501 D111 01 1110105 (PP\/BF)"]}]},{"name":"European Regional Development Fund within the Innovation Economy Operational Program","award":["POIG.02.02.0 0 00 025\/09\/ and POIG.02.01.00 14 122\/09"],"award-info":[{"award-number":["POIG.02.02.0 0 00 025\/09\/ and POIG.02.01.00 14 122\/09"]}]},{"name":"European Regional Development Fund within the Innovation Economy Operational Program","award":["POIG.02.02.0 0 00 025\/09\/ and POIG.02.01.00 14 122\/09"],"award-info":[{"award-number":["POIG.02.02.0 0 00 025\/09\/ and POIG.02.01.00 14 122\/09"]}]},{"name":"European Regional Development Fund within the Innovation Economy Operational Program","award":["POIG.02.02.0 0 00 025\/09\/ and POIG.02.01.00 14 122\/09"],"award-info":[{"award-number":["POIG.02.02.0 0 00 025\/09\/ and POIG.02.01.00 14 122\/09"]}]},{"name":"European Regional Development Fund within the Innovation Economy Operational Program","award":["POIG.02.02.0 0 00 025\/09\/ and POIG.02.01.00 14 122\/09"],"award-info":[{"award-number":["POIG.02.02.0 0 00 025\/09\/ and POIG.02.01.00 14 122\/09"]}]},{"name":"European Regional Development Fund within the Innovation Economy Operational Program","award":["POIG.02.02.0 0 00 025\/09\/ and POIG.02.01.00 14 122\/09"],"award-info":[{"award-number":["POIG.02.02.0 0 00 025\/09\/ and POIG.02.01.00 14 122\/09"]}]},{"name":"European Regional Development Fund within the Innovation Economy Operational Program","award":["POIG.02.02.0 0 00 025\/09\/ and POIG.02.01.00 14 122\/09"],"award-info":[{"award-number":["POIG.02.02.0 0 00 025\/09\/ and POIG.02.01.00 14 122\/09"]}]},{"DOI":"10.13039\/501100012526","name":"Faculty of Chemistry, Warsaw University of Technology","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100012526","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title><jats:p><jats:italic>E. coli<\/jats:italic> purine nucleoside phosphorylase is a homohexamer, which structure, in the apo form, can be described as a trimer of dimers. Earlier studies suggested that ligand binding and kinetic properties are well described by two binding constants and two sets of kinetic constants. However, most of the crystal structures of this enzyme complexes with ligands do not hold the three-fold symmetry, but only two-fold symmetry, as one of the three dimers is different (both active sites in the open conformation) from the other two (one active site in the open and one in the closed conformation). Our recent detailed studies conducted over broad ligand concentration range suggest that protein\u2013ligand complex formation in solution actually deviates from the two-binding-site model. To reveal the details of interactions present in the hexameric molecule we have engineered a single tryptophan Y160W mutant, responding with substantial intrinsic fluorescence change upon ligand binding. By observing various physical properties of the protein and its various complexes with substrate and substrate analogues we have shown that indeed three-binding-site model is necessary to properly describe binding of ligands by both the wild type enzyme and the Y160W mutant. Thus we have pointed out that a symmetrical dimer with both active sites in the open conformation is not forced to adopt this conformation by interactions in the crystal, but most probably the dimers forming the hexamer in solution are not equivalent as well. This, in turn, implies that an allosteric cooperation occurs not only within a dimer, but also among all three dimers forming a hexameric molecule.<\/jats:p>","DOI":"10.1038\/s41598-021-90472-4","type":"journal-article","created":{"date-parts":[[2021,5,27]],"date-time":"2021-05-27T10:16:36Z","timestamp":1622110596000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Single tryptophan Y160W mutant of homooligomeric E. coli purine nucleoside phosphorylase implies that dimers forming the hexamer are functionally not equivalent"],"prefix":"10.1038","volume":"11","author":[{"given":"Marta","family":"Narczyk","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"\u0141ukasz","family":"Mioduszewski","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aleksandra","family":"Oksiejuk","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maria","family":"Winiewska-Szajewska","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Beata","family":"Wielgus-Kutrowska","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Adrian","family":"Gojd\u017a","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joanna","family":"Cie\u015bla","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Agnieszka","family":"Bzowska","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2021,5,27]]},"reference":[{"key":"90472_CR1","doi-asserted-by":"publisher","first-page":"349","DOI":"10.1016\/S0163-7258(00)00097-8","volume":"88","author":"A Bzowska","year":"2000","unstructured":"Bzowska, A., Kulikowska, E. & Shugar, D. Purine nucleoside phosphorylases: properties, functions, and clinical aspects. Pharmacol. Ther. 88, 349\u2013452 (2000).","journal-title":"Pharmacol. Ther."},{"key":"90472_CR2","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1042\/bj3610001","volume":"361","author":"MJ Pugmire","year":"2002","unstructured":"Pugmire, M. J. & Ealick, S. E. Structural analyses reveal two distinct families of nucleoside phosphorylases. Biochem J. 361, 1\u201325 (2002).","journal-title":"Biochem J."},{"key":"90472_CR3","doi-asserted-by":"publisher","first-page":"145","DOI":"10.1177\/1759720X16646703","volume":"8","author":"SE Sattui","year":"2016","unstructured":"Sattui, S. E. & Gaffo, A. L. Treatment of hyperuricemia in gout: current therapeutic options, latest developments and clinical implications. Ther. Adv. Musculoskelet. Dis. 8, 149\u2013149 (2016).","journal-title":"Ther. Adv. Musculoskelet. Dis."},{"key":"90472_CR4","doi-asserted-by":"publisher","first-page":"1173","DOI":"10.1016\/j.chembiol.2003.11.008","volume":"10","author":"EM Bennett","year":"2003","unstructured":"Bennett, E. M. et al. Designer gene therapy using an Escherichia coli purine nucleoside phosphorylase\/prodrug system. Chem. Biol. 10, 1173\u20131181 (2003).","journal-title":"Chem. Biol."},{"key":"90472_CR5","doi-asserted-by":"publisher","first-page":"1348","DOI":"10.1097\/00029330-200708010-00010","volume":"120","author":"JH Zhou","year":"2007","unstructured":"Zhou, J. H., Tang, B., Liu, X. L., He, D. W. & Yang, D. T. hTERT-targeted E. coli purine nucleoside phosphorylase gene\/6-methylpurine deoxyribose therapy for pancreatic cancer. Chin. Med. J. 120, 1348\u20131352 (2007).","journal-title":"Chin. Med. J."},{"key":"90472_CR6","doi-asserted-by":"publisher","first-page":"616","DOI":"10.1016\/j.ejmech.2015.12.029","volume":"108","author":"AE Hassan","year":"2016","unstructured":"Hassan, A. E., Abou-Elkhair, R. A., Parker, W. B. & Allan, P. W. 6-Methylpurine derived sugar modified nucleosides: Synthesis and in vivo antitumor activity in D54 tumor expressing M64V-Escherichia coli purine nucleoside phosphorylase. Eur. J. Med. Chem. 108, 616\u2013622 (2016).","journal-title":"Eur. J. Med. Chem."},{"key":"90472_CR7","doi-asserted-by":"publisher","first-page":"1126","DOI":"10.1002\/jcb.25396","volume":"117","author":"G Cacciapuoti","year":"2016","unstructured":"Cacciapuoti, G. et al. Efficient fludarabine-activating PNP from archaea as a guidance for redesign the active site of E. Coli PNP. J. Cell. Biochem. 117, 1126\u20131135 (2016).","journal-title":"J. Cell. Biochem."},{"key":"90472_CR8","doi-asserted-by":"publisher","first-page":"1373","DOI":"10.1016\/S0969-2126(97)00287-6","volume":"5","author":"C Mao","year":"1997","unstructured":"Mao, C. et al. The crystal structure of Escherichia coli purine nucleoside phosphorylase: A comparison with the human enzyme reveals a conserved topology. Structure 5, 1373\u20131383 (1997).","journal-title":"Structure"},{"key":"90472_CR9","doi-asserted-by":"publisher","first-page":"351","DOI":"10.1006\/jmbi.2001.5211","volume":"315","author":"G Koellner","year":"2002","unstructured":"Koellner, G. et al. Open and closed conformation of the E. coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism. J. Mol. Biol. 315, 351\u2013371 (2002).","journal-title":"J. Mol. Biol."},{"key":"90472_CR10","doi-asserted-by":"publisher","first-page":"117","DOI":"10.5562\/cca2116","volume":"86","author":"Z \u0160tefani\u0107","year":"2013","unstructured":"\u0160tefani\u0107, Z. et al. Still a long way to fully understanding the molecular mechanism of Escherichia coli purine nucleoside phosphorylase. Croat. Chem. Acta 86, 117\u2013127 (2013).","journal-title":"Croat. Chem. Acta"},{"key":"90472_CR11","doi-asserted-by":"publisher","first-page":"377","DOI":"10.1111\/j.1432-1033.1976.tb10031.x","volume":"61","author":"KF Jensen","year":"1976","unstructured":"Jensen, K. F. Purine nucleoside phosphorylase from Salmonella typhimurium and Escherichia coli Initial velocity kinetics, ligand binding, reaction mechanism. Eur. J. Biochem. 61, 377\u2013386 (1976).","journal-title":"Eur. J. Biochem."},{"key":"90472_CR12","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1016\/S0301-4622(96)02239-9","volume":"63","author":"B Kierdaszuk","year":"1997","unstructured":"Kierdaszuk, B., Modrak-W\u00f3jcik, A. & Shugar, D. Binding of phosphate and sulfate anions by purine nucleoside phosphorylase from E. Coli: ligand-dependent quenching of enzyme intrisic fluorescence. Biophys. Chem. 63, 107\u2013118 (1997).","journal-title":"Biophys. Chem."},{"key":"90472_CR13","doi-asserted-by":"publisher","first-page":"1610","DOI":"10.1016\/j.biochi.2011.05.030","volume":"93","author":"G Mikleu\u0161evi\u0107","year":"2011","unstructured":"Mikleu\u0161evi\u0107, G. et al. Validation of the catalytic mechanism of Escherichia coli purine nucleoside phosphorylase by structural and kinetic studies. Biochemie 93, 1610\u20131622 (2011).","journal-title":"Biochemie"},{"key":"90472_CR14","doi-asserted-by":"publisher","first-page":"31","DOI":"10.1016\/j.abb.2018.01.011","volume":"642","author":"B Wielgus-Kutrowska","year":"2018","unstructured":"Wielgus-Kutrowska, B., Grycuk, T. & Bzowska, A. Part-of-the-sites binding and reactivity in the homooligomeric enzymes\u2014Facts and artifacts. Arch. Biochem. Biophys. 642, 31\u201345 (2018).","journal-title":"Arch. Biochem. Biophys."},{"key":"90472_CR15","doi-asserted-by":"publisher","first-page":"1860","DOI":"10.1111\/febs.12746","volume":"281","author":"B Bertosa","year":"2014","unstructured":"Bertosa, B. et al. Homooligomerization is needed for stability: a molecular modelling and solution study of Escherichia coli purine nucleoside phosphorylase. FEBS J. 281, 1860\u20131871 (2014).","journal-title":"FEBS J."},{"key":"90472_CR16","doi-asserted-by":"publisher","first-page":"153","DOI":"10.1006\/jmbi.1998.1799","volume":"280","author":"G Koellner","year":"1998","unstructured":"Koellner, G., Lui\u0107, M., Shugar, D., Saenger, W. & Bzowska, A. Crystal structure of the ternary complex of E. coli purine nucleoside phosphorylase with formycin B, a structural analogue of the substrate inosine, and phosphate (Sulphate) at 2.1 A resolution. J. Mol. Biol. 280, 153\u2013166 (1998).","journal-title":"J. Mol. Biol."},{"key":"90472_CR17","doi-asserted-by":"publisher","first-page":"47110","DOI":"10.1074\/jbc.M304622200","volume":"278","author":"EM Bennet","year":"2003","unstructured":"Bennet, E. M., Li, C., Allan, P. W., Parker, W. B. & Ealick, S. E. Structural basis for substrate specificity of Escherichia coli purine nucleoside phosphorylase. J. Biol. Chem. 278, 47110\u201347118 (2003).","journal-title":"J. Biol. Chem."},{"key":"90472_CR18","doi-asserted-by":"publisher","first-page":"967","DOI":"10.1016\/j.febslet.2012.02.039","volume":"586","author":"Z \u0160tefani\u0107","year":"2012","unstructured":"\u0160tefani\u0107, Z. et al. New phosphate binding sites in the crystal structure of Escherichia coli purine nucleoside phosphorylase complexed with phosphate and formycin A. FEBS Lett. 586, 967\u2013971 (2012).","journal-title":"FEBS Lett."},{"key":"90472_CR19","doi-asserted-by":"publisher","first-page":"15427","DOI":"10.1038\/s41598-018-33723-1","volume":"8","author":"Z \u0160tefani\u0107","year":"2018","unstructured":"\u0160tefani\u0107, Z. et al. Crystallographic snapshots of ligand binding to hexameric purine nucleoside phosphorylase and kinetic studies give insight into the mechanism of catalysis. Sci. Rep. 8, 15427 (2018).","journal-title":"Sci. Rep."},{"key":"90472_CR20","doi-asserted-by":"publisher","first-page":"137","DOI":"10.1016\/j.ymeth.2014.11.012","volume":"76","author":"H Zhao","year":"2015","unstructured":"Zhao, H., Piszczek, G. & Schuck, P. SEDPHAT\u2014A platform for global ITC analysis and global multi-method analysis of molecular interactions. Methods 76, 137\u2013148 (2015).","journal-title":"Methods"},{"key":"90472_CR21","doi-asserted-by":"publisher","first-page":"30","DOI":"10.1110\/ps.062558507","volume":"16","author":"JCD Houtman","year":"2007","unstructured":"Houtman, J. C. D. et al. Studying multi-site binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: Application to adaptor protein complexes in cell signaling. Prot. Sci. 16, 30\u201342 (2007).","journal-title":"Prot. Sci."},{"key":"90472_CR22","doi-asserted-by":"publisher","first-page":"147","DOI":"10.1214\/aoms\/1177731909","volume":"11","author":"A Wald","year":"1940","unstructured":"Wald, A. & Wolfowitz, J. On a test whether two samples are from the same population. Ann. Math. Statist. 11, 147\u2013162 (1940).","journal-title":"Ann. Math. Statist."},{"key":"90472_CR23","volume-title":"Model Selection and Multimodel Inference","author":"KP Burnham","year":"2002","unstructured":"Burnham, K. P. & Anderson, D. R. Model Selection and Multimodel Inference (Springer, 2002)."},{"key":"90472_CR24","doi-asserted-by":"publisher","first-page":"73","DOI":"10.1007\/s13361-015-1239-2","volume":"27","author":"S Kazazi\u0107","year":"2015","unstructured":"Kazazi\u0107, S. et al. New insights into active site conformation dynamics of E. coli PNP revealed by combined H\/D exchange approach and molecular dynamic simulations. J. Am. Soc. Mass. Spectrom. 27, 73\u201382 (2015).","journal-title":"J. Am. Soc. Mass. Spectrom."},{"key":"90472_CR25","doi-asserted-by":"publisher","first-page":"193","DOI":"10.1021\/ja00885a019","volume":"85","author":"JW Jones","year":"1963","unstructured":"Jones, J. W. & Robins, R. K. Purine nucleosides. III. Methylation studies of certain naturally ocurring purine nucleosides. J. Am. Chem. Soc. 85, 193\u2013221 (1963).","journal-title":"J. Am. Chem. Soc."},{"key":"90472_CR26","doi-asserted-by":"publisher","first-page":"115","DOI":"10.1016\/0076-6879(66)08014-5","volume":"8","author":"BN Ames","year":"1966","unstructured":"Ames, B. N. Assay for inorganic phosphate, total phosphate and phosphonates. Meth. Enzymol. 8, 115\u2013118 (1966).","journal-title":"Meth. Enzymol."},{"key":"90472_CR27","doi-asserted-by":"publisher","first-page":"180","DOI":"10.1006\/prep.2001.1437","volume":"22","author":"J Lee","year":"2001","unstructured":"Lee, J. et al. Expression, purification, and characterization of recombinant purine nucleoside phosphorylase from Escherichia coli. Protein. Expr. Purif. 22, 180\u2013188 (2001).","journal-title":"Protein. Expr. Purif."},{"key":"90472_CR28","doi-asserted-by":"publisher","first-page":"429","DOI":"10.1016\/S0021-9258(17)30997-3","volume":"167","author":"HM Kalckar","year":"1947","unstructured":"Kalckar, H. M. Differential spectrophotometry of purine compounds by means of specific enzymes. J. Biol. Chem. 167, 429\u2013443 (1947).","journal-title":"J. Biol. Chem."},{"key":"90472_CR29","doi-asserted-by":"publisher","first-page":"355","DOI":"10.1016\/0167-4838(86)90035-X","volume":"874","author":"E Kulikowska","year":"1986","unstructured":"Kulikowska, E., Bzowska, A., Wierzchowski, J. & Shugar, D. Properties of two unusual, and fluorescent, substrates of purine-nucleoside phosphorylase: 7-methylguanosine and 7-methylinosine. Biochim. Biophys. Acta. 874, 355\u2013363 (1986).","journal-title":"Biochim. Biophys. Acta."},{"key":"90472_CR30","volume-title":"Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems","author":"IH Segel","year":"1993","unstructured":"Segel, I. H. Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems (Wiley-Interscience, 1993)."},{"key":"90472_CR31","unstructured":"Laue, T. M. et al.\u00a0Computer-aided interpretation of sedimentation data for proteins. In\u00a0Analytical Ultracentrifugation in Biochemistry and Polymer Science (eds. Harding S. E., Rowe, A. J. & Horton, J. C.) 90\u2013125 (Royal Society of Chemistry, Cambridge 1992)."},{"key":"90472_CR32","doi-asserted-by":"publisher","first-page":"1606","DOI":"10.1016\/S0006-3495(00)76713-0","volume":"78","author":"P Schuck","year":"2000","unstructured":"Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and Lamm equation modeling. Biophys. J. 78, 1606\u20131619 (2000).","journal-title":"Biophys. J."},{"key":"90472_CR33","doi-asserted-by":"publisher","first-page":"125","DOI":"10.1107\/S0907444909047337","volume":"66","author":"W Kabsch","year":"2010","unstructured":"Kabsch, W. XDS. A. Cryst. D 66, 125\u2013132 (2010).","journal-title":"A. Cryst. D"},{"key":"90472_CR34","doi-asserted-by":"publisher","first-page":"658","DOI":"10.1107\/S0021889807021206","volume":"40","author":"AJ McCoy","year":"2007","unstructured":"McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Cryst. 40, 658\u2013674 (2007).","journal-title":"J. Appl. Cryst."},{"key":"90472_CR35","doi-asserted-by":"publisher","first-page":"352","DOI":"10.1107\/S0907444912001308","volume":"68","author":"PV Afonine","year":"2012","unstructured":"Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. A. Cryst. D 68, 352\u2013367 (2012).","journal-title":"A. Cryst. D"},{"key":"90472_CR36","doi-asserted-by":"publisher","first-page":"2126","DOI":"10.1107\/S0907444904019158","volume":"60","author":"P Emsley","year":"2004","unstructured":"Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. A. Cryst. D 60, 2126\u20132132 (2004).","journal-title":"A. Cryst. D"},{"key":"90472_CR37","doi-asserted-by":"publisher","first-page":"571","DOI":"10.1385\/1-59259-890-0:571","volume-title":"The Proteomics Protocols Handbook","author":"E Gasteiger","year":"2005","unstructured":"Gasteiger, E. et al. Protein identification and analysis tools on the ExPASy server. In The Proteomics Protocols Handbook (ed. Walker, J. M.) 571\u2013607 (Humana Press, 2005)."},{"key":"90472_CR38","doi-asserted-by":"publisher","first-page":"1747","DOI":"10.1111\/j.1742-4658.2010.07598.x","volume":"277","author":"K Breer","year":"2010","unstructured":"Breer, K. et al. 9-Deazaguanine derivatives connected by a linker to difluoromethylene phosphonic acid are slow-binding picomolar inhibitors of trimeric purine nucleoside phosphorylase. FEBS J. 277, 1747\u20131760 (2010).","journal-title":"FEBS J."},{"key":"90472_CR39","unstructured":"CRC Handbook for Chemistry and Physics. (100th Edition, CRC Press, 2019)."},{"key":"90472_CR40","doi-asserted-by":"publisher","first-page":"260","DOI":"10.1006\/abio.1996.0238","volume":"237","author":"P Kuzmic","year":"1996","unstructured":"Kuzmic, P. Program DYNAFIT for the analysis of enzyme kinetic data: Application to HIV proteinase. Anal. Biochem. 237, 260\u2013273 (1996).","journal-title":"Anal. Biochem."},{"key":"90472_CR41","doi-asserted-by":"publisher","first-page":"124","DOI":"10.1016\/j.ymeth.2014.11.018","volume":"76","author":"CA Brautigan","year":"2015","unstructured":"Brautigan, C. A. Fitting two- and three-site binding models to isothermal titration calorimetric data. Methods 76, 124\u2013136 (2015).","journal-title":"Methods"},{"key":"90472_CR42","doi-asserted-by":"publisher","first-page":"101","DOI":"10.1016\/j.molstruc.2014.03.009","volume":"1077","author":"M Jerabek-Willemsen","year":"2014","unstructured":"Jerabek-Willemsen, M. et al. MicroScale Thermophoresis: Interaction analysis and beyond. J. Mol. Str. 1077, 101\u2013133 (2014).","journal-title":"J. Mol. Str."},{"key":"90472_CR43","first-page":"5066","volume":"84","author":"S Keller","year":"2012","unstructured":"Keller, S. et al. High-precision isothermal titration calorimetry with automated peak-shape analysis. Anal. Biochem. 84, 5066\u20135073 (2012).","journal-title":"Anal. Biochem."},{"key":"90472_CR44","doi-asserted-by":"publisher","first-page":"1627","DOI":"10.1021\/ac60214a047","volume":"36","author":"A Savitzky","year":"1964","unstructured":"Savitzky, A. & Golay, M. J. E. Smoothing and differentiation of data by simplified least squares procedures. Anal. Chem. 36, 1627\u20131639 (1964).","journal-title":"Anal. Chem."}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-90472-4.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-90472-4","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-90472-4.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,3]],"date-time":"2022-12-03T08:52:23Z","timestamp":1670057543000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-021-90472-4"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,5,27]]},"references-count":44,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2021,12]]}},"alternative-id":["90472"],"URL":"https:\/\/doi.org\/10.1038\/s41598-021-90472-4","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,5,27]]},"assertion":[{"value":"1 February 2021","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"26 April 2021","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"27 May 2021","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"11144"}}