bb35f6aca2055f52f1589a1f281c5193 270634.pdf c9ebdebf86191131884127444f7406c0bb4aabe1 270634.pdf 9541ab441e60deb98baebc48f5e0dfc750cdee0c2b9e65e09ff5035944eb89a6 270634.pdf Title: Stabilization Effect of Intrinsically Disordered Regions on Multidomain Proteins: The Case of the Methyl-CpG Protein 2, MeCP2 Subject: Intrinsic disorder plays an important functional role in proteins. Disordered regions are linked to posttranslational modifications, conformational switching, extra/intracellular trafficking, and allosteric control, among other phenomena. Disorder provides proteins with enhanced plasticity, resulting in a dynamic protein conformational/functional landscape, with well-structured and disordered regions displaying reciprocal, interdependent features. Although lacking well-defined conformation, disordered regions may affect the intrinsic stability and functional properties of ordered regions. MeCP2, methyl-CpG binding protein 2, is a multifunctional transcriptional regulator associated with neuronal development and maturation. MeCP2 multidomain structure makes it a prototype for multidomain, multifunctional, intrinsically disordered proteins (IDP). The methyl-binding domain (MBD) is one of the key domains in MeCP2, responsible for DNA recognition. It has been reported previously that the two disordered domains flanking MBD, the N-terminal domain (NTD) and the intervening domain (ID), increase the intrinsic stability of MBD against thermal denaturation. In order to prove unequivocally this stabilization effect, ruling out any artifactual result from monitoring the unfolding MBD with a local fluorescence probe (the single tryptophan in MBD) or from driving the protein unfolding by temperature, we have studied the MBD stability by differential scanning calorimetry (reporting on the global unfolding process) and chemical denaturation (altering intramolecular interactions by a different mechanism compared to thermal denaturation). Keywords: MeCP2; intrinsically disordered proteins; structural stability; thermal and chemical denaturation; differential scanning calorimetry Author: David Ortega-Alarcon, Rafael Claveria-Gimeno, Sonia Vega, Olga C. Jorge-Torres, Manel Esteller, Olga Abian and Adrian Velazquez-Campoy Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Mon Aug 16 11:26:56 2021 CEST ModDate: Mon Aug 16 11:30:35 2021 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 18 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 2716166 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- EIFDAO+VnURWPalladioL Type 1 Custom yes yes no 10 0 EFMFIQ+URWPalladioL-Ital Type 1 Custom yes yes no 15 0 ZMPEUX+URWPalladioL-Bold Type 1 Custom yes yes no 20 0 LLLWZV+URWPalladioL-Roma Type 1 Custom yes yes no 24 0 IVVSWS+TimesNewRomanPS-BoldItalicMT Type 1C WinAnsi yes yes no 34 0 MCBCCI+SourceSansRoman-Regular Type 1C Custom yes yes no 64 0 MCBCCM+SourceSansRoman.613wght Type 1C Custom yes yes no 68 0 MCBCCO+SourceSansRoman-Bold Type 1C Custom yes yes 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Title: Materials and Methods Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7b69c6ba Children: Item: Title: Protein Expression and Purification Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@46daef40 Item: Title: Double-Stranded DNA Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@12f41634 Item: Title: Thermal Denaturation Assessment by Differential Scanning Calorimetry Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@13c27452 Item: Title: Chemical Denaturation Assessment by Fluorescence Spectroscopy Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@262b2c86 Item: Title: Dynamic Light Scattering (DLS) Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@371a67ec Item: Title: Results Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@5ed828d Children: Item: Title: The MBD Is Stabilized against Thermal Unfolding by Its Disordered Flanking Domains Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@50d0686 Item: Title: The MBD Is Stabilized against Chemical Unfolding by Its Disordered Flanking Domains Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@7a3d45bd Item: Title: MBD Molecular Size Is Highly Susceptible to the Presence of the Flanking Domains Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1e7c7811 Item: Title: Discussion Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@77ec78b9 Item: Title: Conclusions Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1a3869f4 Item: Title: References Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@a38d7a3 Info: Title: Stabilization Effect of Intrinsically Disordered Regions on Multidomain Proteins: The Case of the Methyl-CpG Protein 2, MeCP2 Author: David Ortega-Alarcon, Rafael Claveria-Gimeno, Sonia Vega, Olga C. Jorge-Torres, Manel Esteller, Olga Abian and Adrian Velazquez-Campoy Subject: Intrinsic disorder plays an important functional role in proteins. Disordered regions are linked to posttranslational modifications, conformational switching, extra/intracellular trafficking, and allosteric control, among other phenomena. Disorder provides proteins with enhanced plasticity, resulting in a dynamic protein conformational/functional landscape, with well-structured and disordered regions displaying reciprocal, interdependent features. Although lacking well-defined conformation, disordered regions may affect the intrinsic stability and functional properties of ordered regions. MeCP2, methyl-CpG binding protein 2, is a multifunctional transcriptional regulator associated with neuronal development and maturation. MeCP2 multidomain structure makes it a prototype for multidomain, multifunctional, intrinsically disordered proteins (IDP). The methyl-binding domain (MBD) is one of the key domains in MeCP2, responsible for DNA recognition. It has been reported previously that the two disordered domains flanking MBD, the N-terminal domain (NTD) and the intervening domain (ID), increase the intrinsic stability of MBD against thermal denaturation. In order to prove unequivocally this stabilization effect, ruling out any artifactual result from monitoring the unfolding MBD with a local fluorescence probe (the single tryptophan in MBD) or from driving the protein unfolding by temperature, we have studied the MBD stability by differential scanning calorimetry (reporting on the global unfolding process) and chemical denaturation (altering intramolecular interactions by a different mechanism compared to thermal denaturation). 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