{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,4,16]],"date-time":"2026-04-16T08:42:39Z","timestamp":1776328959536,"version":"3.50.1"},"reference-count":44,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2021,6,18]],"date-time":"2021-06-18T00:00:00Z","timestamp":1623974400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100010198","name":"Ministerio de Econom\u00eda, Industria y Competitividad, Gobierno de Espa\u00f1a","doi-asserted-by":"publisher","award":["BFU2016-78232-P and PID2019-107293GB-I00"],"award-info":[{"award-number":["BFU2016-78232-P and PID2019-107293GB-I00"]}],"id":[{"id":"10.13039\/501100010198","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000780","name":"European Commission","doi-asserted-by":"publisher","award":["Interreg-SUDOE (Grant Neuromed)"],"award-info":[{"award-number":["Interreg-SUDOE (Grant Neuromed)"]}],"id":[{"id":"10.13039\/501100000780","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100011100","name":"Fundaci\u00f3n Espa\u00f1ola para la Ciencia y la Tecnolog\u00eda","doi-asserted-by":"publisher","award":["PRECIPITA"],"award-info":[{"award-number":["PRECIPITA"]}],"id":[{"id":"10.13039\/501100011100","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100010067","name":"Gobierno de Arag\u00f3n","doi-asserted-by":"publisher","award":["LMP30_18 and E45_17R"],"award-info":[{"award-number":["LMP30_18 and E45_17R"]}],"id":[{"id":"10.13039\/501100010067","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Human phenylalanine hydroxylase (PAH) is a metabolic enzyme involved in the catabolism of L-Phe in liver. Loss of conformational stability and decreased enzymatic activity in PAH variants result in the autosomal recessive disorder phenylketonuria (PKU), characterized by developmental and psychological problems if not treated early. One current therapeutic approach to treat PKU is based on pharmacological chaperones (PCs), small molecules that can displace the folding equilibrium of unstable PAH variants toward the native state, thereby rescuing the physiological function of the enzyme. Understanding the PAH folding equilibrium is essential to develop new PCs for different forms of the disease. We investigate here the urea and the thermal-induced denaturation of full-length PAH and of a truncated form lacking the regulatory and the tetramerization domains. For either protein construction, two distinct transitions are seen in chemical denaturation followed by fluorescence emission, indicating the accumulation of equilibrium unfolding intermediates where the catalytic domains are partly unfolded and dissociated from each other. According to analytical centrifugation, the chemical denaturation intermediates of either construction are not well-defined species but highly polydisperse ensembles of protein aggregates. On the other hand, each protein construction similarly shows two transitions in thermal denaturation measured by fluorescence or differential scanning calorimetry, also indicating the accumulation of equilibrium unfolding intermediates. The similar temperatures of mid denaturation of the two constructions, together with their apparent lack of response to protein concentration, indicate the catalytic domains are unfolded in the full-length PAH thermal intermediate, where they remain associated. That the catalytic domain unfolds in the first thermal transition is relevant for the choice of PCs identified in high throughput screening of chemical libraries using differential scanning fluorimetry.<\/jats:p>","DOI":"10.3390\/ijms22126539","type":"journal-article","created":{"date-parts":[[2021,6,18]],"date-time":"2021-06-18T11:19:20Z","timestamp":1624015160000},"page":"6539","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Unravelling the Complex Denaturant and Thermal-Induced Unfolding Equilibria of Human Phenylalanine Hydroxylase"],"prefix":"10.3390","volume":"22","author":[{"given":"Mar\u00eda","family":"Conde-Gim\u00e9nez","sequence":"first","affiliation":[{"name":"Departamento de Bioqu\u00edmica y Biolog\u00eda Molecular y Celular, Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Units: BIFI-IQFR (CSIC) and GBsC-CSIC, University of Zaragoza, 50009 Zaragoza, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2879-9200","authenticated-orcid":false,"given":"Javier","family":"Sancho","sequence":"additional","affiliation":[{"name":"Departamento de Bioqu\u00edmica y Biolog\u00eda Molecular y Celular, Biocomputation and Complex Systems Physics Institute (BIFI)-Joint Units: BIFI-IQFR (CSIC) and GBsC-CSIC, University of Zaragoza, 50009 Zaragoza, Spain"},{"name":"Aragon Health Research Institute (IIS Arag\u00f3n), 50009 Zaragoza, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,6,18]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"341","DOI":"10.1002\/iub.1150","article-title":"Phenylalanine hydroxylase: Function, structure, and regulation","volume":"65","author":"Flydal","year":"2013","journal-title":"IUBMB Life"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1417","DOI":"10.1016\/S0140-6736(10)60961-0","article-title":"Phenylketonuria","volume":"376","author":"Blau","year":"2010","journal-title":"Lancet"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"302","DOI":"10.1038\/ejhg.2014.114","article-title":"Linking genotypes database with locus-specific database and genotype\u2013phenotype correlation in phenylketonuria","volume":"23","author":"Wettstein","year":"2014","journal-title":"Eur. 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