000109534 001__ 109534
000109534 005__ 20230519145623.0
000109534 0247_ $$2doi$$a10.3390/ijms22126539
000109534 0248_ $$2sideral$$a126718
000109534 037__ $$aART-2021-126718
000109534 041__ $$aeng
000109534 100__ $$0(orcid)0000-0003-4358-9110$$aConde-Giménez, María
000109534 245__ $$aUnravelling the Complex Denaturant and Thermal-Induced Unfolding Equilibria of Human Phenylalanine Hydroxylase
000109534 260__ $$c2021
000109534 5060_ $$aAccess copy available to the general public$$fUnrestricted
000109534 5203_ $$aHuman 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.
000109534 536__ $$9info:eu-repo/grantAgreement/ES/DGA/E45-17R$$9info:eu-repo/grantAgreement/ES/MICINN/PID2019-107293GB-I00$$9info:eu-repo/grantAgreement/ES/MINECO/BFU2016-78232-P
000109534 540__ $$9info:eu-repo/semantics/openAccess$$aby$$uhttp://creativecommons.org/licenses/by/3.0/es/
000109534 590__ $$a6.208$$b2021
000109534 592__ $$a1.176$$b2021
000109534 594__ $$a6.9$$b2021
000109534 591__ $$aBIOCHEMISTRY & MOLECULAR BIOLOGY$$b69 / 297 = 0.232$$c2021$$dQ1$$eT1
000109534 593__ $$aComputer Science Applications$$c2021$$dQ1
000109534 591__ $$aCHEMISTRY, MULTIDISCIPLINARY$$b50 / 180 = 0.278$$c2021$$dQ2$$eT1
000109534 593__ $$aInorganic Chemistry$$c2021$$dQ1
000109534 593__ $$aSpectroscopy$$c2021$$dQ1
000109534 593__ $$aOrganic Chemistry$$c2021$$dQ1
000109534 593__ $$aPhysical and Theoretical Chemistry$$c2021$$dQ1
000109534 593__ $$aMolecular Biology$$c2021$$dQ1
000109534 655_4 $$ainfo:eu-repo/semantics/article$$vinfo:eu-repo/semantics/publishedVersion
000109534 700__ $$0(orcid)0000-0002-2879-9200$$aSancho, Javier$$uUniversidad de Zaragoza
000109534 7102_ $$11002$$2060$$aUniversidad de Zaragoza$$bDpto. Bioq.Biolog.Mol. Celular$$cÁrea Bioquímica y Biolog.Mole.
000109534 773__ $$g22, 12 (2021), 22126539 [17 pp.]$$pInt. j. mol. sci.$$tInternational Journal of Molecular Sciences$$x1661-6596
000109534 8564_ $$s3498985$$uhttps://zaguan.unizar.es/record/109534/files/texto_completo.pdf$$yVersión publicada
000109534 8564_ $$s2815373$$uhttps://zaguan.unizar.es/record/109534/files/texto_completo.jpg?subformat=icon$$xicon$$yVersión publicada
000109534 909CO $$ooai:zaguan.unizar.es:109534$$particulos$$pdriver
000109534 951__ $$a2023-05-18-16:18:07
000109534 980__ $$aARTICLE