biomolecules Article Stabilization Effect of Intrinsically Disordered Regions on Multidomain Proteins: The Case of the Methyl-CpG Protein 2, MeCP2 David Ortega-Alarcon 1 , Rafael Claveria-Gimeno 1,2,3,† , Sonia Vega 1 , Olga C. Jorge-Torres 4 , Manel Esteller 4,5,6,7 , Olga Abian 1,2,3,8,9, * and Adrian Velazquez-Campoy 1,3,8,9,10, * 1 Institute of Biocomputation and Physics of Complex Systems (BIFI), Joint Units IQFR-CSIC-BIFI, and GBsC-CSIC-BIFI, Universidad de Zaragoza, 50018 Zaragoza, Spain; dortega@bifi.es (D.O.-A.); rafacg@certest.es (R.C.-G.); svega@bifi.es (S.V.) 2 Instituto Aragonés de Ciencias de la Salud (IACS), 50009 Zaragoza, Spain 3 Instituto de Investigación Sanitaria de Aragón (IIS Aragón), 50009 Zaragoza, Spain 4 Josep Carreras Leukaemia Research Institute (IJC), 08916 Badalona, Spain; ojorge@carrerasresearch.org (O.C.J.-T.); mesteller@carrerasresearch.org (M.E.) 5 Centro de Investigacion Biomedica en Red Cancer (CIBERONC), 28029 Madrid, Spain 6 Institucio Catalana de Recerca i Estudis Avançats (ICREA), 08010 Barcelona, Spain 7 Physiological Sciences Department, School of Medicine and Health Sciences, University of Barcelona (UB),  l’Hospitalet de Llobregat, 08907 Barcelona, Spain  8 Centro de Investigación Biomédica en Red en el Área Temática de Enfermedades Hepáticas y Digestivas (CIBERehd), 28029 Madrid, Spain Citation: Ortega-Alarcon, D.; 9 Departamento de Bioquímica y Biología Molecular y Celular, Claveria-Gimeno, R.; Vega, S.; Universidad de Zaragoza, 50009 Zaragoza, Spain Jorge-Torres, O.C.; Esteller, M.; Abian, 10 Fundación ARAID, Gobierno de Aragón, 50009 Zaragoza, Spain O.; Velazquez-Campoy, A. * Correspondence: oabifra@unizar.es (O.A.); adrianvc@unizar.es (A.V.-C.); Stabilization Effect of Intrinsically Tel.: +34-876-555417 (O.A.); +34-976-762996 (A.V.-C.) Disordered Regions on Multidomain † Current address: Certest Biotec S.L., 50840 Zaragoza, Spain. Proteins: The Case of the Methyl-CpG Protein 2, MeCP2. Biomolecules 2021, Abstract: Intrinsic disorder plays an important functional role in proteins. Disordered regions are 11, 1216. https://doi.org/10.3390/ linked to posttranslational modifications, conformational switching, extra/intracellular trafficking, biom11081216 and allosteric control, among other phenomena. Disorder provides proteins with enhanced plasticity, resulting in a dynamic protein conformational/functional landscape, with well-structured and Academic Editors: Simona disordered regions displaying reciprocal, interdependent features. Although lacking well-defined Maria Monti, Giuseppina De Simone conformation, disordered regions may affect the intrinsic stability and functional properties of and Emma Langella ordered regions. MeCP2, methyl-CpG binding protein 2, is a multifunctional transcriptional regulator associated with neuronal development and maturation. MeCP2 multidomain structure makes it Received: 10 July 2021 Accepted: 13 August 2021 a prototype for multidomain, multifunctional, intrinsically disordered proteins (IDP). The methyl- Published: 16 August 2021 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 Publisher’s Note: MDPI stays neutral domain (NTD) and the intervening domain (ID), increase the intrinsic stability of MBD against with regard to jurisdictional claims in thermal denaturation. In order to prove unequivocally this stabilization effect, ruling out any published maps and institutional affil- artifactual result from monitoring the unfolding MBD with a local fluorescence probe (the single iations. 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). Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Keywords: MeCP2; intrinsically disordered proteins; structural stability; thermal and chemical This article is an open access article denaturation; differential scanning calorimetry distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Biomolecules 2021, 11, 1216. https://doi.org/10.3390/biom11081216 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 1216 2 of 18 1. Introduction Intrinsically disordered proteins (IDP) and intrinsically disordered regions (IDR) are involved in many physiological mechanisms and pathologies. Although much effort has been dedicated towards understanding their structural and functional properties, IDPs and IDRs remain largely elusive. IDPs and IDRs contain a large proportion of polar, hydrophilic residues, compared to well-folded proteins and regions, which provide them the capability of populating a dynamic, rich ensemble of structures [1,2]. They do not fold spontaneously into a stable conformation in aqueous solvent because they lack a hydrophobic core or it is insufficiently large, but they are usually very susceptible to environmental factors (e.g., presence of ligands and interacting macromolecules, redox state, or pH of the surroundings) and posttranslational modifications [3]. Interestingly, they may fold into well-defined conformations when interacting with a biological partner or under appropriate conditions (the binding partner or concomitant processes, such as electron transfer or de/protonation, provide the additional physico-chemical context for establishing stabilizing interactions and triggering the disorder-to-order transition) or remain disordered (“fuzzy” complexes). Disorder in proteins provides some unexpected advantages. First, disorder increases the structural plasticity and flexibility, allowing proteins to adopt multiple conformations within a complex conformational landscape depending on the environmental conditions, which is important for interacting with many biological partners [4,5] (in fact, IDPs or proteins containing IDRs represent important hubs in metabolic and signaling networks) and undergoing conformational changes required for cellular trafficking, internalization, and degradation [6,7]. Second, disorder makes some regions more susceptible to post- translational modifications [8], which represents another protein regulation level condition- ing the accessible conformations and the potential interactions with other biomolecules. Thirdly, disorder endows proteins with an additional regulation level based on allosteric control [9–12], which broadly consists in the regulation of the conformational landscape by ligand interaction (where ligand is any molecule interacting with a given protein: ion, small molecule, macromolecule.) [13,14]. Protein disorder has been maintained and exploited by evolution [15–18]. Therefore, it must be a key property that must be understood and accounted for when investigating structural and functional features in proteins. Some consequences derived from the pres- ence of IDRs and the direct connection between order/disorder and function in proteins are not fully understood yet. Disordered regions in particular contribute to protein stability and function, sometimes with a counterbalancing effect [19,20], and should be considered as functional regions and not just flexible stretches [21,22]. The methyl-CpG binding protein 2, MeCP2, a multidomain protein containing a large proportion of intrinsic disorder and interacting with many biological partners, is a physiologically interesting and clinically relevant IDP. MeCP2 is involved in many key physiological processes associated with neuronal development, maturation, and plas- ticity [23,24], and MeCP2 deleterious mutations are associated with Rett syndrome, a neurodevelopmental disorder related to the autistic spectrum [25–27]. Although all do- mains must be relevant, because MeCP2 is a main transcriptional regulator and chromatin remodeling element, two domains emerge among the others: the methyl-binding domain (MBD) and the transcriptional repression domain (TRD). MBD is the domain responsible for the interaction with methylated-CpG-rich promoters [28–30]. Interestingly, MBD has a DNA binding site and contains one of the few well-structured regions in this protein, which is flanked by two completely disordered domains: N-terminal domain (NTD) and in- tervening domain (ID). Despite their lack of structure, both domains are critical for MeCP2 functions. In particular, mutations and posttranslational modifications in NTD affect and modulate MeCP2 functions [31,32]. In addition, the two identified MeCP2 isoforms differ in just a few residues located at the beginning of the NTD, but that small difference has important consequences in function, expression, and structural properties [33]. The ID Biomolecules 2021, 11, 1216 3 of 18 not only provides a second DNA binding site, but it also considerably increases the DNA binding affinity of the MBD site. It has been reported that both NTD and ID increase the intrinsic structural stability of MBD, as observed by thermal unfolding experiments: at pH 7, the unfolding temperature Tm is 38.4 ◦ C and 46.2 ◦ C for MBD and NTD-MBD-ID, respectively, and the unfolding en- thalpy ∆Hm is 38 kcal/mol and 46 kcal/mol for MBD and NTD-MBD-ID, respectively [34]. All other domains also have a minor stabilizing effect on MBD [35]. These unfolding experiments were undertaken by following the intrinsic fluorescence emission of the sin- gle tryptophan located at the MBD (W104), and consequently they accurately reflect the thermal stability of MBD. This stabilization effect is an intriguing outcome, considering that NTD and ID do not adopt well-defined structures by themselves. There are two main explanations for this phenomenon: (1) NTD and ID, although disordered or with high susceptibility to populate unfolded structures, are able to interact specifically with MBD or unspecifically (e.g., fuzzy interactions) (e.g., through long range polar/electrostatic interac- tions due to their large content in polar residues), thus increasing the intrinsic stability of MBD against unfolding; and (2) NTD and ID, being disordered and populating a multitude of different unfolded structures, may exert a steric hindrance effect on MBD, promote its compaction, and restrict its early expansion close to the unfolding temperature of the isolated MBD. It might be speculated that the observed apparent stabilization effect exerted by NTD and ID could be some kind of artifact when determining the apparent stability of MBD due to: (1) using the intrinsic fluorescence of W104 as a local probe just reporting an unfolding process restricted to the vicinity of that residue; or (2) using temperature as a physico-chemical stress for altering the noncovalent interatomic interactions responsible for maintaining the folded MBD structure and, thus, triggering the protein unfolding process. In fact, a slightly destabilizing effect for NTD and ID on MBD was reported before [36], but minor differences in the protein constructs and/or experimental conditions could explain that disagreement. To rule out those possibilities, we have employed differential scanning calorimetry in order to use a thermal unfolding technique providing a global signal with contribution from the entire protein molecule, and we have also monitored the unfolding process by performing chemical denaturations using denaturant concentration as the physico-chemical stress triggering the protein unfolding process. Two MeCP2 constructions have been employed: the isolated MBD, and the MBD together with its two flanking domains, NTD-MBD-ID. The overall conclusion is that both completely disordered domains, NTD and ID, unequivocally stabilize the MBD against thermal and chemical denaturation. Therefore, disorder in proteins may be considered a pervasive feature that plays an important role in many the allosteric control of protein conformation, protein interactions, and protein regulation (modifications, trafficking, degradation, in/activation.). This may be even more important in multidomain IDPs with a complex conformational and multifunctional landscape [13,14]. 2. Materials and Methods 2.1. Protein Expression and Purification Protein variants (MBD and NTD-MBD-ID) were expressed and purified following identical procedures. Plasmids (pET30b) containing both constructions were transformed into BL21 (DE3) Star E. coli strain. Starting cultures were grown in 150 mL of LB/kanamycin (50 µg/mL) at 37 ◦ C overnight. Then, 4 L of LB/kanamycin (25 µg/mL) were inoculated (1:100 dilution) and incubated under the same conditions until reaching an optical density (λ = 600 nm) of 0.6. Protein expression was induced by adding isopropyl 1-thio-β-D- galactopyranoside (IPTG) 1 mM at 18 ◦ C overnight. Cells were sonicated in ice and benzonase (Merck-Millipore, Madrid, Spain) was added (20 U/mL) to remove nucleic acids. Proteins were purified using metal affinity chromatography using a HiTrap TALON column (GE-Healthcare Life Sciences, Barcelona, Spain) with two washing steps: buffer sodium phosphate 50 mM, pH 7, NaCl 300 mM, Biomolecules 2021, 11, 1216 4 of 18 and buffer sodium phosphate 50 mM, pH 7, NaCl 800 mM. Elution was performed applying an imidazole 10–150 mM elution gradient. Protein purity was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The polyhistidine-tag was removed by processing with GST-tagged PreScission Pro- tease in proteolytic cleavage buffer (Tris-HCl 50 mM, NaCl 150 mM, pH 7.5) at 4 ◦ C for 4 h. Progress of the proteolytic processing was monitored by SDS-PAGE. The protein was further purified with a combination of two affinity chromatographic steps for removing the polyhistidine-tag (HiTrap TALON column) and the GST-tagged PreScission Protease (GST TALON column, from GE-Healthcare Life Sciences, Barcelona, Spain). Purity and homo- geneity were evaluated by SDS-PAGE and size-exclusion chromatography. Storage buffer consisted of Tris 50 mM pH 7.0 and pooled samples were kept at −80 ◦ C. The identity of all proteins was checked by mass spectrometry (4800plus MALDI-TOF/MS, from Applied Biosystems-Thermo Fisher scientific, Waltham, MA, USA). Potential DNA contamination was always estimated by UV absorption 260/280 ratio. Because a single tryptophan is located in MBD, an extinction coefficient of 11,460 M−1 cm−1 at 280 nm was employed for the two variants. 2.2. Double-Stranded DNA HPLC-purified methylated and unmethylated 45-bp single-stranded DNA (ssDNA) oligomers, corresponding to the promoter IV of the mouse brain-derived neurotrophic factor (BDNF) gene [36,37], were purchased from Integrated DNA Technologies (Leuven, Belgium). Two complementary pairs of ssDNA were used for DNA binding assays: forward unmethylated: 50 -GCCATGCCCTGGAACGGAACTCTCCTAATAAAAG-ATGT ATCATTT-30 ; reverse unmethylated: 50 -AAATGATACATCTTTTATTAGGAGAGTTCCGTTCC-AGGGCA TGGC-30 ; forward mCpG: 50 -GCCATGCCCTGGAA(5-Me)CGGAACTCTCCTAATAAA-AGATGTA TCATTT-30 ; reverse mCpG: 50 -AAATGATACATCTTTTATTAGGAGAGTTC(5-Me)CGTT-CCAGGGCA TGGC-30 . The ssDNA oligonucleotides were dissolved at a concentration of 0.5 mM and the concentration was assessed by using the extinction coefficient provided by the manufacturer. Complementary ssDNA oligomers were mixed at an equimolar ratio and annealed to obtain 45-bp double-stranded DNA (dsDNA) using a Stratagene Mx3005P qPCR real-time thermal cycler (Agilent Technologies, Santa Clara, CA, USA). The thermal annealing profile consisted of: (1) equilibration at 25 ◦ C for 30 s; (2) heating ramp up to 99 ◦ C; (3) equilibration at 99 ◦ C for 1 min; and (4) 3 h cooling process down to 25 ◦ C at a rate of 1 ◦ C/3 min. 2.3. Thermal Denaturation Assessment by Differential Scanning Calorimetry Thermal stability of MeCP2 MBD and NTD-MBD-ID was assessed by temperature unfolding monitored by high-precision differential scanning calorimetry (DSC). The partial molar heat capacity of the protein in solution was measured as a function of temperature in an Auto-PEAQ-DSC (MicroCal, Malvern-Panalytical, Malvern, UK). Experiments were performed with a protein solution at a concentration of 20–40 µM in Tris 50 mM pH 7.0, and scanning from 15 to 95 ◦ C at a scan rate of 60 ◦ C/h. The first approach in DSC experimental analysis consisted of applying a model-free data analysis for discriminating between different possibilities: two-state unfolding, non- two-state unfolding, and oligomer unfolding of the protein. From the thermogram (excess molar heat capacity of the protein as a function of the temperature, ∆CP (T)), the calorimetric unfolding enthalpy, ∆Hcal , the unfolding temperature, Tm , and the maximal unfolding heat capacity, CP,max , were estimated, from which it was possible to calculate the van’t Hoff enthalpy, ∆HvH : 4RTm 2C P,max ∆HvH = (1) ∆Hcal Biomolecules 2021, 11, 1216 5 of 18 From the ratio ∆HvH /∆Hcal different possibilities may arise: (1) if ∆HvH /∆Hcal = 1, the thermogram would reflect a single transition and the protein unfolds according to a two- state model (i.e., the protein contains a single energetic domain); (2) if ∆HvH /∆Hcal < 1, the thermogram would reflect, at least, two (partially) overlapping transitions and the protein unfolds according to a non-two-state model (i.e., the protein contains, at least, two domains which unfold in an independent manner); and (3) if ∆HvH /∆Hcal > 1, the thermogram would reflect an unfolding transition coupled to subunit dissociation (i.e., the protein is oligomeric and unfolds into monomers). Once the appropriate unfolding model can be selected according to the previous van‘t Hoff test, the thermogram was analyzed by non-linear regression fitting analysis consider- ing a set of n independent transitions, each characterized by a transition temperature Tm,i , an unfolding enthalpy ∆Hi (Tm,i ), and an unfolding heat capacity ∆CP,i : n Ki ( T ) ∆Hi ( T )2 h∆CP ( T )i = ∑ 2 RT 2 i =1 (1+Ki ( T )) (2)  ∆Gi ( T ) Ki ( T) = exp(− /RT )  ∆Gi ( T ) = ∆Hi ( Tm,i ) 1 − T Tm,i + ∆CP,i T − Tm,i − T ln TTm,i where Ki , ∆Hi , and ∆Gi are the equilibrium constant, the unfolding enthalpy, and the stabilization Gibbs energy for the protein conformational transition i, respectively, R is the ideal gas constant, and T is the absolute temperature. 2.4. Chemical Denaturation Assessment by Fluorescence Spectroscopy Chemical stability of MeCP2 MBD and NTD-MBD-ID was assessed by fluorescence spectroscopy, using ultra-pure urea (urea crystalline pharma grade, PanReac, Barcelona, Spain) as chaotropic denaturant. The intrinsic fluorescence emission of the single tryp- tophan in MeCP2 (W104) was monitored inn a thermostated Cary Eclipse fluorescence spectrophotometer (Varian-Agilent, Santa Clara, CA, USA) using a protein concentration of 5 µM, in a 1 cm path-length quartz cuvette (Hellma Analytics, Müllheim, Germany). Experiments were performed in Tris 50 mM pH 7.0, at 20 ◦ C, with temperature controlled by a Peltier unit. Fluorescence emission spectra were recorded from 300 to 500 nm, using an excitation wavelength of 290 nm and a bandwidth of 5 nm, at different urea concentrations ([D] = 0–7 M, with increments of 0.25 M). Samples were equilibrated overnight at room temperature before measurements. Guanidinium hydrochloride was avoided because it is a charged denaturant and would interfere with electrostatic interactions between protein charges and it is known to be a stabilizing molecule at low concentration [38,39]. Consider- ing only the native and the unfolded states of the protein (two-state model) was sufficient for reproducing the denaturation curves, and inclusion of additional intermediate partially unfolded states was detrimental for the goodness of the fitting. Three unfolding-reporting signals were employed for quantitative analysis. The fluorescence emission intensity I at a single wavelength (in the case of MeCP2, 340 nm, i.e., I340 ), or the intensity ratio at two wavelengths (in the case of MeCP2, I330 /I350 ) as a function of denaturant concentration, which, for an unfolding process involving independent transitions (corresponding to in- dependently unfolding regions or domains), must be analyzed according to this set of equations: K ([ D ]) I ([ D ]) = 1+K1([ D]) IN ([ D ]) + 1+K ([ D]) IU ([ D ]) K ([ D ]) = exp(−∆G ([ D ])/RT ) (3) ∆G ([ D ]) = ∆Gw ([ D ]) − m[ D ] Ii ([ D ]) = Ai + Bi [ D ] where K, ∆G, ∆Gw, and m are the equilibrium constant, the stabilization Gibbs energy in the presence and the absence of denaturant, and the susceptibility of ∆G to the denaturant concentration, for the conformational transition. The linear extrapolation model [40,41] has been assumed when accounting for the effect of denaturant concentration [D] on the stabilization energy, and the dependence of the intrinsic fluorescence intensity of each Biomolecules 2021, 11, 1216 6 of 18 protein conformational state with the denaturant concentration was considered to be linear. The average emission energy of the spectrum as a function of denaturant concentration, which must be analyzed in a similar way (Equation (3)), was also considered: ∑i Ii /λi h Ei = (4) ∑i Ii where Ii is the fluorescence emission intensity in the spectrum at a certain wavelength λi . Thus, is the spectral average value of the inverse of the wavelength. The advantage of using the fluorescence intensity is that is more quantitatively rigor- ous. However, the fluorescence intensity focuses on a certain region of the spectrum, while the average energy adds the possibility of taking into account changes along the entire spectrum for quantifying global spectral changes. Other unfolding observables, such as the wavelength for maximal intensity may lack proportionality with the advance of the unfolding process, may not change significantly upon unfolding, or may make the analysis more complex [42,43]. When needed, methylated and unmethylated dsDNA were added at 6 µM in order to test the ability of the protein to interact with dsDNA and evaluate the extent of the stabilization induced by DNA binding under the same conditions. 2.5. Dynamic Light Scattering (DLS) Dynamic light scattering measurements were performed in a DynaPro Plate Reader III (Wyatt Technology, Santa Barbara, CA, USA) using a 384-multiwell plate (Aurora Microplates, Whitefish, MT, USA). Urea stock solution (8 M) was serially diluted to obtain concentrations ranging from 0 M to 7 M (steps of 1 M), before the addition of protein to a final concentration of 20 µM. All solutions were filtered using 0.2-µm membranes and protein stocks were centrifuged in microtubes for 2 min at maximum speed to prevent urea precipitation or protein aggregates from interfering with the DLS measurements. For each measurement, 10 acquisitions of 3 s were taken, and the apparent hydrodynamic radius was estimated from the experimental diffusion coefficient, obtained by the cumulant fit of the translational autocorrelation function, assuming a Rayleigh sphere model. Experiments were performed in Tris 50 mM pH 7.0, at 20 ◦ C. 3. Results 3.1. The MBD Is Stabilized against Thermal Unfolding by Its Disordered Flanking Domains The DSC thermograms showed a single apparent unfolding transition for MBD and NTD-MBD-ID (Figure 1). Experiments performed at 20 and 40 µM protein concentrations provided similar results (similar apparent Tm and unfolding enthalpy values), and therefore the native protein remains monomeric and the unfolding Biomolecules 2021, 11, x FOR PEER REVIEW 7 of 18 does not trigger oligomerization or aggregation. Figure 1. Differential scanning calorimetry thermograms (excess molar heat capacity of the protein as aFigure function of1. Differential temperature) for MBDscanning calorimetry (squares) and NTD-MBD-ID thermograms (circles). Experiments were (excess done molar heat capacity of the protein withasa aconcentration functionof of 40 µM, in Tris 50 mM, pH temperature) 7. Non-linear for fittings are shown MBD (squares) and(red), according NTD-MBD-ID (circles). Experiments were done to a single transition model (for MBD) and a two independent transitions model (for NTD-MBD- with a concentration of 40 µM, in Tris 50 mM, pH 7. Non-linear fittings are shown (red), according to ID). a single transition The model-free model van‘t Hoff analysis(for MBD) indicated that and a two asindependent MBD unfolds transitions a single transition, model (for NTD-MBD-ID). whereas NTD-MBD-ID unfolds through two independent transitions, because the en- thalpies ratio is close to 1 for MBD, but rather lower than 1 for NTD-MBD-ID (Table 1). The thermal unfolding monitored by fluorescence (using the single tryptophan W104 as an intrinsic probe) was previously analyzed according to a single transition (two-state model) and provided unfolding parameters in reasonable agreement with those reported now: transition temperature of 38.4 and 46.2 °C for MBD and NTD-MBD-ID, respectively, Biomolecules 2021, 11, 1216 7 of 18 The model-free van‘t Hoff analysis indicated that MBD unfolds as a single transition, whereas NTD-MBD-ID unfolds through two independent transitions, because the enthalpies ratio is close to 1 for MBD, but rather lower than 1 for NTD-MBD-ID (Table 1). The thermal unfolding monitored by fluorescence (using the single tryptophan W104 as an intrinsic probe) was previously analyzed according to a single transition (two-state model) and provided unfolding parameters in reasonable agreement with those reported now: transition temperature of 38.4 and 46.2 ◦ C for MBD and NTD-MBD-ID, respectively, and unfolding enthalpy of 29 and 37 kcal/mol for MBD and NTD-MBD-ID, respectively. Thus, it seems that the presence of the two disordered flanking domains increased the stability of MBD and the unfolding process of the longer construct occurred with unfolding intermediates. Table 1. Parameters associated with the van ‘t Hoff test (or calorimetric two-state test). Protein ∆H cal CPmax ∆H vH T m (◦ C) Ratio Construction (kcal/mol) (kcal/K·mol) (kcal/mol) MBD 37.8 36 1.85 39 1.08 NTD-MBD-ID 44.8 52 2.73 42 0.81 According to these results, the MBD unfolding was analyzed with a model considering a single transition and that for NTD-MBD-ID was analyzed with a model considering two independent transitions (Figure 1 and Table 2). Table 2. Thermal unfolding parameters for MBD and NTD-MBD-ID at pH 7. Protein ∆H 1 ∆H 2 Sqrt(RSS) T m1 (◦ C) T m2 (◦ C) Construction (kcal/mol) (kcal/mol) (kcal/mol) MBD 37.4 ± 0.2 38 ± 1 – – 1.0603 45.0 ± 0.2 47 ± 1 – – 2.0606 NTD-MBD-ID 41.7 ± 0.2 11 ± 1 45.0 ± 0.2 46 ± 1 1.5540 Sqrt(RSS): square root of the residual sum of squares. Although the unfolding of NTD-MBD-ID could be reasonably fitted with a single transition model, the residuals sum of squares (RSS) was smaller for the model with two transitions. More rigorously, the parametric F-test indicated that the model considering two transitions is statistically more appropriate (F = 50.40 > F3213 (α = 0.05) = 2.65). To observe other potential differences between MBD and NTD-MBD-ID, and to compare stabilities under different physico-chemical stresses, fluorescence chemical denaturations using urea as chaotropic denaturant were performed. 3.2. The MBD Is Stabilized against Chemical Unfolding by Its Disordered Flanking Domains Because there is a single tryptophan residue (W104) in the whole MeCP2 sequence, and it is located in the MBD, measuring the intrinsic fluorescence of this residue is a good indicator of the folding state of MBD in both constructions. In the absence of denaturant, both protein constructs showed non-symmetrical, bell-shaped spectra, with a maximum around 340 nm indicating that W104 was not exposed to the solvent (Figure 2). As the concentration of denaturant increased, the intensity of emission was dramatically reduced (quenching) and red-shifted, with a maximum near 350 nm, as the fraction of unfolded protein increased, indicating that W104 was exposed to the solvent. indicator of the folding state of MBD in both constructions. In the absence of denaturant, both protein constructs showed non-symmetrical, bell-shaped spectra, with a maximum around 340 nm indicating that W104 was not exposed to the solvent (Figure 2). As the concentration of denaturant increased, the intensity of emission was dramatically reduced Biomolecules 2021, 11, 1216 (quenching) and red-shifted, with a maximum near 350 nm, as the fraction of unfolded 8 of 18 protein increased, indicating that W104 was exposed to the solvent. MBD + CpG-dsDNA MBD + mCpG-dsDNA MBD 1000 1000 1000 800 800 800 600 600 600 400 400 400 200 200 200 0 0 0 300 350 400 450 500 300 350 400 450 500 300 350 400 450 500 Wavelength (nm) Wavelength (nm) Wavelength (nm) NTD-MBD-ID NTD-MBD-ID + CpG-dsDNA NTD-MBD-ID + mCpG-dsDNA 1000 1000 1000 800 800 800 600 600 600 400 400 400 200 200 200 0 0 0 300 350 400 450 500 300 350 400 450 500 300 350 400 450 500 Wavelength (nm) Wavelength (nm) Wavelength (nm) Figure 2. Raw fluorescence spectra for MBD (top) and NTD-MBD-ID (bottom) at different urea concentrations ([D] = Figure 2. Raw fluorescence spectra for MBD (top) and NTD-MBD-ID (bottom) at different urea concentrations ([D] = 0–7 0–7 M) in the absence of dsDNA (left), in the presence of unmethylated CpG-dsDNA (middle), and methylated mCpG- M) in the absence of dsDNA (left), in the presence of unmethylated CpG-dsDNA (middle), and methylated mCpG-dsDNA dsDNA (right). (right). Protein stabilization upon dsDNA was also determined by fluorescence chemical Protein stabilization upon dsDNA was also determined by fluorescence chemical de- denaturation assays. When dsDNA was present, the emitted fluorescence intensity was naturation assays. When dsDNA was present, the emitted fluorescence intensity was con- considerably diminished due to light absorption by dsDNA at these wavelengths. Raw spec- siderably Biomolecules 2021, 11, x FOR PEER REVIEW diminished tra were processed indue to to order light absorption calculate by dsDNA the spectral at these average wavelengths. energy Raw spec- 9 of 18 at each experimental tra were processed condition, in order totraces and the unfolding calculate werethe spectral average constructed (Figureenergy 3). at each experimental condition, and the unfolding traces were constructed (Figure 3). Figure3.3.Chemical Figure Chemicalunfolding unfoldingcurves curvesfor forMBD MBD(circles) (circles)and andNTD-MBD-ID NTD-MBD-ID(squares). (squares).The Thespectral spectral average energy of the protein was represented as a function of the urea concentration: free protein average energy of the protein was represented as a function of the urea concentration: free protein (black), protein bound to unmethylated CpG-dsDNA (blue), and methylated mCpG-dsDNA (black), protein bound to unmethylated CpG-dsDNA (blue), and methylated mCpG-dsDNA (red). (red). Thedisordered The disorderedflanking flankingdomains domainsexerted exertedaasubtle subtlestabilizing stabilizingeffect effecton onMBD, MBD,as asseen seen inthe in thechemical chemicalunfolding unfolding curves curvesmonitoring monitoring the thespectral spectralaverage averageenergy energy(Figure (Figure33andand Table3). Table 3).The Thebinding bindingofofdsDNA dsDNAalways alwaysproduced producedstabilization stabilizationof ofMBD MBDagainst againstchemical chemical denaturation denaturation(Figure (Figure33and andTable Table3). 3). Table 3. Thermal unfolding parameters for MBD and NTD-MBD-ID at pH 7, estimated by by ana- lyzing the denaturant dependence of the spectral average energy. Protein DNA ΔGw (kcal/mol) m (kcal/mol·M) [D]1/2 (M) Construction -- 2.5 0.77 3.3 MBD CpG-dsDNA 2.7 0.68 3.9 Biomolecules 2021, 11, 1216 9 of 18 Table 3. Thermal unfolding parameters for MBD and NTD-MBD-ID at pH 7, estimated by by analyzing the denaturant dependence of the spectral average energy. Protein DNA ∆Gw (kcal/mol) m (kcal/mol·M) [D]1/2 (M) Construction – 2.5 0.77 3.3 MBD CpG-dsDNA 2.7 0.68 3.9 mCpG-dsDNA 3.2 0.67 4.8 – 2.8 0.70 4.0 NTD-MBD-ID CpG-dsDNA 4.2 0.72 5.9 mCpG-dsDNA 7.6 0.97 7.8 [D]1/2 is the half-unfolding denaturant concentration. The stabilization Gibbs energy in the absence of denaturant is related to [D]1/2 : ∆Gw = m [D]1/2 . The stabilizing effect was dramatically increased when the dsDNA was present. It must be born in mind that the presence of ID not only increases 400-fold the dsDNA binding affinity (from micromolar affinity to nanomolar affinity), but also provides an additional binding dsDNA site with micromolar affinity (the extent of the stabilization effect depends on the stoichiometry of the interaction and the binding affinity, among other factors) [34]. The interaction with mCpG-dsDNA was strikingly stabilizing for the protein. Indeed, even at high urea concentrations (i.e., [D] > 6 M), a large fraction of the protein (> 50%) seemed to remain folded. Unfolding traces could be satisfactorily analyzed employing an unfolding model with a single transition, and the unfolding parameters were estimated by non-linear least-squares regression analysis (Table 3). For comparison, the spectral series shown in Figure 2 were also analyzed focusing Biomolecules 2021, 11, x FOR PEER REVIEW fluorescence intensity at a single wavelength (340 nm), as shown in Figure on the 10 4ofand 18 Table 4, and the intensity ratio at two wavelengths (to reduce potential uncertainty and variability due to the protein concentration), as shown in Figure 5 and Table 5. Figure4. Figure Chemical unfolding 4. Chemical unfolding curves curvesfor forMBD MBD(circles) and (circles) andNTD-MBD-ID NTD-MBD-ID (squares). TheThe (squares). fluorescence fluores- cence intensity of the protein at 340nm was represented as a function of the urea concentration: free intensity of the protein at 340nm was represented as a function of the urea concentration: free protein protein (black), protein bound to unmethylated CpG-dsDNA (blue), and methylated (black), protein bound to unmethylated CpG-dsDNA (blue), and methylated mCpG-dsDNA (red). mCpG- dsDNA (red). Table 4. Thermal unfolding parameters for MBD and NTD-MBD-ID at pH 7, estimated by analyzing the denaturant dependence of the fluorescence intensity at 340 nm. Protein DNA ΔGw (kcal/mol) m (kcal/mol·M) [D]1/2 (M) Construction -- 2.3 0.84 2.7 MBD CpG-dsDNA 2.7 0.92 2.9 mCpG-dsDNA 2.9 0.83 3.5 -- 2.7 0.78 3.4 NTD-MBD-ID CpG-dsDNA 3.6 0.64 5.6 Figure 4. Chemical unfolding curves for MBD (circles) and NTD-MBD-ID (squares). The fluores- Biomolecules 2021, 11, 1216 10 free cence intensity of the protein at 340nm was represented as a function of the urea concentration: of 18 protein (black), protein bound to unmethylated CpG-dsDNA (blue), and methylated mCpG- dsDNA (red). Table 4. Table Thermalunfolding 4.Thermal unfoldingparameters parameters for forMBD MBDand andNTD-MBD-ID NTD-MBD-ID at atpH pH7,7,estimated estimatedby byanalyzing analyzing thedenaturant the denaturantdependence dependenceofofthe thefluorescence fluorescence intensity intensity at at 340 340 nm. nm. Protein Protein DNA DNA ∆G ΔGww(kcal/mol) (kcal/mol) mm(kcal/mol ·M) (kcal/mol·M) [D] [D] (M) 1/21/2 (M) Construction Construction – 2.3 0.84 2.7 -- 2.3 0.84 2.7 MBD CpG-dsDNA 2.7 0.92 2.9 MBD CpG-dsDNA 2.7 0.92 2.9 mCpG-dsDNA 2.9 0.83 3.5 mCpG-dsDNA 2.9 0.83 3.5 – 2.7 0.78 3.4 -- 2.7 0.78 3.4 NTD-MBD-ID CpG-dsDNA 3.6 0.64 5.6 NTD-MBD-ID CpG-dsDNA 3.6 0.64 5.6 mCpG-dsDNA 3.8 0.49 7.7 mCpG-dsDNA 3.8 0.49 7.7 [D] is the half-unfolding denaturant concentration. The stabilization Gibbs energy in the absence of denaturant 1/2 [D]1/2 is the half-unfolding denaturant concentration. The stabilization Gibbs energy in the absence is related to [D]1/2 : ∆Gw = m [D]1/2 . of denaturant is related to [D]1/2: ΔGw = m [D]1/2. Figure 5. Figure Chemicalunfolding 5. Chemical unfoldingcurves curvesfor for MBD MBD (circles) (circles) and and NTD-MBD-ID NTD-MBD-ID (squares). (squares). The The ratio ratio of of fluorescence fluorescenceintensities intensitiesatat330 330and and350 350nmnmwas wasrepresented representedas asaafunction functionof ofthe theurea ureaconcentration: concentration: free freeprotein protein(black), (black),protein protein bound bound to to unmethylated unmethylatedCpG-dsDNA CpG-dsDNA(blue), (blue),and andmethylated methylatedmCpG- mCpG- dsDNA (red). dsDNA (red). Table 5. Thermal unfolding parameters for MBD and NTD-MBD-ID at pH 7, estimated by analyzing the denaturant dependence of the fluorescence intensity ratio I330 /I350 . Protein DNA ∆Gw (kcal/mol) m (kcal/mol·M) [D]1/2 (M) Construction – 3.6 1.02 3.5 MBD CpG-dsDNA 3.9 1.04 3.7 mCpG-dsDNA 4.8 1.06 4.6 – 4.1 1.09 3.8 NTD-MBD-ID CpG-dsDNA 6.9 1.24 5.6 mCpG-dsDNA 7.7 1.10 7.0 [D]1/2 is the half-unfolding denaturant concentration. The stabilization Gibbs energy in the absence of denaturant is related to [D]1/2 : ∆Gw = m [D]1/2 . 3.3. MBD Molecular Size Is Highly Susceptible to the Presence of the Flanking Domains The apparent hydrodynamic radius of the two constructs, MBD and NTD-MBD-ID, was measured under different conditions: different urea concentrations and in the ab- sence/presence of dsDNA (Figure 6 and Table 6). The size histograms showed average and standard deviation values that were modulated by urea concentration (Supplementary of denaturant is related to [D]1/2: ΔGw = m [D]1/2. 3.3. MBD Molecular Size Is Highly Susceptible to the Presence of the Flanking Domains The apparent hydrodynamic radius of the two constructs, MBD and NTD-MBD-ID, Biomolecules 2021, 11, 1216 was measured under different conditions: different urea concentrations and in the11ab- of 18 sence/presence of dsDNA (Figure 6 and Table 6). The size histograms showed average and standard deviation values that were modulated by urea concentration (Supplemen- tary Figure S2). In addition, we could observe a similar effect from unmethylated and Figure S2). In addition, we could observe a similar effect from unmethylated and methy- methylated dsDNA. lated dsDNA. Figure Figure6.6.Apparent Apparent hydrodynamic hydrodynamic radius radiusofof MBD MBDand andNTD-MBD-ID NTD-MBD-ID measured measured bybyDLS DLSofofMBD MBD (empty (emptycircles) and circles) NTD-MBD-ID and NTD-MBD-ID (squares) in the (squares) absence in the of DNA absence (black), of DNA in theinpresence (black), of un-of the presence methylated unmethylatedCpG-dsDNA (blue), CpG-dsDNA andand (blue), the the presence presenceof of methylated mCpG-dsDNA methylated mCpG-dsDNA (red). (red).Control Control measurements of dsDNA are also shown (gray circles). measurements of dsDNA are also shown (gray circles). Table 6. Hydrodynamic radius of the two protein constructs in complex with dsDNA estimated by Table 6. Hydrodynamic radius of the two protein constructs in complex with dsDNA estimated by dynamic light scattering. dynamic light scattering. Protein construction DNA Radius (nm) Protein Construction DNA Radius (nm) -- 1.6 MBD – CpG-dsDNA 1.6 3.7 MBD CpG-dsDNA 3.7 mCpG-dsDNA mCpG-dsDNA 3.8 3.8 -- 5.5 – 5.5 NTD-MBD-ID NTD-MBD-ID CpG-dsDNA CpG-dsDNA 4.3 4.3 mCpG-dsDNA mCpG-dsDNA 3.9 3.9 -- – CpG-dsDNA CpG-dsDNA 3.5 3.5 In the absence of urea, MBD showed a hydrodynamic radius close to that predicted from its molecular weight. As the concentration of urea increased, the apparent hydrody- namic radius of MBD increased, as expected for an unfolding process, but the apparent hydrodynamic radius of NTD-MBD-ID decreased. This unexpected result may be related to the large proportion of disorder, the large proportion of charged residues, and the increase in dielectric constant of the solvent when urea concentration increases, as discussed later. The interpretation of the low susceptibility and small increase of the apparent hydrody- namic radius of the two constructs to the concentration of urea in the presence of dsDNA is difficult, because it must be a combination of several effects: initial stabilization of protein by dsDNA interaction is weakened because of the reduction in the dielectric constant of the solvent (weakened polar/electrostatic interactions between protein and dsDNA) and the preferential interaction of urea with the protein residues displacing water molecules, leading to dsDNA dissociation and protein destabilization and unfolding. 4. Discussion The impact of disordered regions on the stability and functional features of well-folded regions in proteins remains as an important and elusive matter intimately connected with protein function regulation and allosteric control. There are some cases where this issue gets especially important. For example, the stabilization induced by intrinsically disordered regions in the HIV-1 Rev protein has been reported [21]. In another example, stability Biomolecules 2021, 11, 1216 12 of 18 changes have been described for nucleoplasmin depending on the length of the disordered tail in each subunit of the pentameric protein: a fifty-residue C-terminal deletion mutant showed lower thermal stability, whereas an eighty C-terminal deletion mutant showed higher thermal stability than the full-length protein [22]. In the case of MeCP2, it has been previously outlined that the two flanking domains of MBD in MeCP2 (NTD and ID) increase MBD stability and dsDNA binding affinity. This is not a simple issue, because those two domains are fully disordered and MBD is also 40% disordered, approximately. The comparison of the circular dichroism spectra for both constructions reflected a lower level of structural order for NTD-MBD-ID (Supplementary Figure S1). The stabilization effect was observed through fluorescence thermal denaturations, and two reasonable objections could be pointed out: (1) the lack of a signal reporting global effects in the protein, because fluorescence intensity only reports the local effect in the surroundings of the single tryptophan in MBD in the thermal denaturations, and (2) the thermal stress driving the unfolding process that could result in an artifactual observation, whereas other unfolding processes (e.g., chemical or pressure denaturation) might provide a somewhat different outcome. In order to rule out these possibilities, we studied the unfolding of MBD and NTD-MBD-ID by differential scanning calorimetry and by chemical denaturation. Both constructions, MBD and NTD-MBD-ID, showed a single apparent transition in their DSC thermogram. Applying the van ‘t Hoff (two-state) test, it could be observed that, although MBD seemed to unfold through a single transition, the unfolding of NTD-MBD- ID involved at least two intermediate unfolding states that can be significantly populated at moderate temperatures. Thus, the conformational landscape of NTD-MBD-ID is more complex than expected, and its unfolding cooperativity is lower than that of MBD. The two unfolding transitions observed in NTD-MBD-ID might correspond to two independent transitions within MBD (the presence of the flanking domains decouples two regions and lowers the unfolding cooperativity of MBD) or might reflect a transition in NTD or ID corresponding to a region that adopts a folded conformation when accompanying the MBD and undergoes unfolding. The stabilization effect exerted by NTD and ID was evident from the comparison of the apparent transition temperatures for both constructs determined by DSC, namely 37.4 ◦ C for MBD and 45 ◦ C for NTD-MBD-ID, which are fairly similar to those determined by fluorescence thermal denaturations (38.4 ◦ C for MBD and 46.2 ◦ C for NTD-MBD-ID). However, a striking phenomenon can be noticed with a further analysis. From the un- folding parameters reported in Table 2, the molar fractions of the relevant conformational states for MBD and NTD-MBD-ID at any temperature can be outlined (Figure 7). It can be observed that at 20 ◦ C, 96% of MBD remains in its native conformation, and the tempera- ture for the unfolded state to be 50% populated was 37.4 ◦ C (the transition temperature). From that, a stabilization Gibbs energy at 20 ◦ C of 1.9 kcal/mol could be estimated, in fair agreement with the 1.4 kcal/mol estimated by thermal fluorescence denaturations. However, although the completely unfolded state of NTD-MBD-ID reaches a 50% pop- ulation at 50.7 ◦ C, the native state is populated only 75% at 20 ◦ C with a corresponding stabilization Gibbs energy of just 0.6 kcal/mol, because of the coexistence of partially unfolded states. Therefore, it is obvious that NTD-MBD-ID requires higher temperatures for complete unfolding compared to MBD, but at low temperature the relevant stability (the stabilization energy gap connecting the native state and the first partially unfolded state) [44] is lower than that of MBD (0.6 kcal/mol for NTD-MBD-ID and 1.9 kcal/mol for MBD), and the integrity of the NTD-MBD-ID native state is compromised to a larger extent at low temperatures. For example, at 37.4 ◦ C (a temperature at which the stabilization Gibbs energy for MBD is zero and both native state and unfolded state are 50% populated), the stabilization Gibbs energy of NTD-MBD-ID is –0.7 kcal/mol and the native state is only 46% populated. bility (the stabilization energy gap connecting the native state and the first partially un- folded state) [44] is lower than that of MBD (0.6 kcal/mol for NTD-MBD-ID and 1.9 kcal/mol for MBD), and the integrity of the NTD-MBD-ID native state is compromised to a larger extent at low temperatures. For example, at 37.4 °C (a temperature at which the Biomolecules 2021, 11, stabilization 1216 Gibbs energy for MBD is zero and both native state and unfolded state are 13 of 18 50% populated), the stabilization Gibbs energy of NTD-MBD-ID is –0.7 kcal/mol and the native state is only 46% populated. Figure 7. Molar fraction Figure or population 7. Molar fraction or (Pi ) of the different population (Pi) of theconformational states: native different conformational state states: (black), native statefirst intermediate (black), first intermediate state (red), second intermediatestate state(red), second (green), intermediate and fully unfoldedstate state(green), andMBD (blue), for fully(left) unfolded state and for (blue), for (right). NTD-MBD-ID MBDcalculated Populations were (left) and with for NTD-MBD-ID the parameters (right). shownPopulations in Table 2. were calculated with the parameters shown in Table 2. The lack of correlation between the apparent Tm ’s and the stabilization Gibbs energy at The lacklow of correlation temperature between the apparent is an indication of theTcaveats m’s andassociated the stabilization Gibbs with using energy only Tm ’s for assessing at low temperature is anstability. structural indication of the caveats A rigorous associated stability with must assessment using involve only Tm’sthefordetermination as- of sessing structural stability.Gibbs stabilization A rigorous energies stability assessment at relevant mustThis conditions. involve the determina- problem parallels with the often- observed tion of stabilization lackenergies Gibbs of correlation between at relevant the half-unfolding conditions. This problem denaturant concentration parallels with the [D]1/2 often-observed andlack the of stabilization correlationGibbsbetween the ∆G energy w , as discussed half-unfolding below. concentration denaturant Temperature [D]1/2 and the stabilization Gibbs and chemical energy ΔGwdenaturation , as discussedmodulate below. and alter intramolecular noncova- lent interactions Temperature and chemical (e.g., hydrogen bonds, denaturation modulateelectrostatic and alterinteractions and salt intramolecular bridges, hydropho- noncova- bic interactions) by different mechanisms. Temperature alters lent interactions (e.g., hydrogen bonds, electrostatic interactions and salt bridges, hydro- the thermal motion, lowers phobic interactions) by different mechanisms. Temperature alters the thermal motion,dielectric con- the barrier to overcome the stabilizing interatomic energies, and reduces the stant, to lowers the barrier with an overall overcome thedestabilizing effect [45]. Urea stabilizing interatomic increases energies, and the dielectric reduces constant (higher the die- than lectric constant, water) with [46], diminishing an overall destabilizingthe effect strength ofUrea [45]. polar/charge increasesinteractions, the dielectricand interacts specif- con- stant (higher ically with the than water) protein [46], backbone diminishing theand displaces strength water molecules of polar/charge by preferential interactions, and interaction loweringwith interacts specifically the the hydration proteincapability backboneof water, and destabilizing displaces the nativeby water molecules state, and shifting the prefer- conformational equilibrium towards (partially and completely) ential interaction lowering the hydration capability of water, destabilizing the native state, unfolded states, with an overall destabilizing effect. The different effect of temperature and shifting the conformational equilibrium towards (partially and completely) unfolded on the dielectric constant (and therefore, on the strength of electrostatic interactions) states, with an overall destabilizing effect. The different effect of temperature on the die- is relevant, since IDPs such as MeCP2 lectric constant contain aon (and therefore, considerable the strength percentage of polar/charged of electrostatic interactions)residues, and specific and is relevant, since IDPs such as MeCP2 contain a considerable percentage of polar/charged residues, electrostatic unspecific electrostatic interactions may be established intramolecularly. Thus, interactions, and specific and unspecific either specific interactions electrostatic direct interactions may beorestablished long-rangeintramolecularly. unspecific interactions, surely play an important role mediating the reciprocal effect between MBD and both flanking domains NTD and ID. An interesting observation arises from the fact that the stabilization effect is considerably diminished at pH 7 when the ionic strength is increased: at NaCl 150 mM, the unfolding temperature Tm is 46.4 ◦ C and 49.8 ◦ C for MBD and NTD-MBD-ID, respectively, and the unfolding enthalpy ∆Hm is 32 kcal/mol and 38 kcal/mol for MBD and NTD-MBD-ID, respectively. IDPs and IDRs are rich in polar/charged residues, and the ionic screening on electrostatic charges will affect both the specific and the unspecific mechanisms of interdependence between folded and unstructured domains. The chemical denaturation of MBD and NTD-MBD-ID followed by fluorescence seem to occur as a single transition. From the experimental data, a clear stabilization effect of NTD and ID can be observed from the apparent half-unfolding denaturant concentration: [D]1/2 is 3.30 M for MBD and 3.95 for NTD-MBD-ID. However, when calculating the stabilization Gibbs energy ∆Gw , the difference is not as clear, and the changes observed in [D]1/2 be obscured in ∆Gw because of the m values. A similar phenomenon occurs when calculating stabilization Gibbs energies at low temperature from the Tm , in which the unfolding heat capacity and enthalpy values are critical for a correct extrapolation. There- fore, estimating stabilities just focusing on Tm or [D]1/2 is risky and limited; stabilization energies at a reference experimental condition (e.g., 20 ◦ C and absence of denaturant) must be determined when assessing structural stability. Biomolecules 2021, 11, 1216 14 of 18 As previously reported, focusing on different observable signals may result in consider- able differences in estimated stability parameters from spectroscopic unfolding curves [42]. In the case of MeCP2, it seems that the analysis based on the fluorescence intensity at 340 nm underestimates the stabilization energies for NTD-MBD-ID in the presence of dsDNA, but the corresponding unfolding traces are ill-defined in the post-transition region because of the high stability. Moreover, the analysis based on the fluorescence intensity ratio seems to overestimate stabilization energies for most of the cases. In this respect, it must be born in mind that the intensity ratio might lack proportionality with the advance of the unfolding process [47,48]. Although the absolute values of stabilization energies do not completely agree, the differences in stabilization energies between protein constructs are rather independent of the signal employed; in particular, the presence of NTD and ID increases the stability of MBD by 0.4 kcal/mol. Thus, even considering the present limitations, the reported data confirm the stabilizing effect of the disordered domains, NTD and ID, on the stability of MBD. The previously reported stabilization Gibbs energies against thermal denaturation [34] are similar to those determined from chemical denaturation, but they are not in full agree- ment. At 20 ◦ C, MBD showed a stabilization energy of 1.4 kcal/mol determined from fluorescence thermal denaturations, while it showed a stabilization energy of 2.5 kcal/mol determined by chemical denaturation. Moreover, NTD-MBD-ID showed a stabilization energy of 2.5 kcal/mol determined from fluorescence thermal denaturations, while it showed a stabilization energy of 2.8 determined by chemical denaturation. Of course, those quantities do not necessarily must be in agreement, because, although the initial state in both unfolding processes is the same (the native state), the final state may be different: the unfolded state after thermal denaturation may be structurally different from the unfolded state after chemical denaturation [49,50]. The same occurs with the unfolding process itself, which may proceed through different routes and involving different inter- mediate states depending on whether temperature or denaturant concentration drives the unfolding process. Regarding the DLS data, MBD showed a hydrodynamic radius similar to that pre- dicted by size-scaling based on molecular weight, but NTD-MBD-ID showed a much larger hydrodynamic radius than the predicted one [51,52]. If MBD were completely folded or unfolded, the hydrodynamic radius would be expected to be around 1.9 nm or 2.9 nm, respectively, and the observed radius is 1.6 nm; therefore, MBD must contain a consid- erable percentage of folded structure, as expected. However, NTD-MBD-ID showed an abnormally large radius (5.5 nm), much larger than that predicted even if fully unfolded. If NTD-MBD-ID were completely folded or unfolded, the hydrodynamic radius would be expected to be around 2.5 nm or 4.6 nm. Therefore, assuming the agreement is reason- able (considering the experimental uncertainties and important approximations applied when estimating the apparent hydrodynamic radius by DLS, as well as the uncertainties associated with estimating the size from the protein molecular weight), NTD-MBD-ID must contain a very large proportion of disorder. It is interesting to point out that the completely unfolded NTD is expected to have a hydrodynamic radius of around 2.6 nm, and the completely unfolded ID is expected to have a hydrodynamic radius of around 1.8 nm. Figure 8 shows the hydrophobicity profile of NTD-MBD-ID, which shows two hydrophobic regions, at the end of NTD and the second half of MBD. Disorder predictions (by DISOPRED3 and IUPRED2 algorithms [53,54]) reveal that structural order is confined to most of MBD, and NTD and ID may remain mostly unstructured. MBD-ID must contain a very large proportion of disorder. It is interesting to point out that the completely unfolded NTD is expected to have a hydrodynamic radius of around 2.6 nm, and the completely unfolded ID is expected to have a hydrodynamic radius of around 1.8 nm. Figure 8 shows the hydrophobicity profile of NTD-MBD-ID, which shows two hydrophobic regions, at the end of NTD and the second half of MBD. Disorder pre- Biomolecules 2021, 11, 1216 15 of 18 dictions (by DISOPRED3 and IUPRED2 algorithms [53,54]) reveal that structural order is confined to most of MBD, and NTD and ID may remain mostly unstructured. Figure8.8. (Left) Figure (Left) Hydrophobicity Hydrophobicity (Kyte (Kyte&&Doolittle Doolittlescale) scale)plot plotforfor NTD-MBD-ID NTD-MBD-ID (black) calculated (black) calculated using Expasy ProtScale tool (https://web.expasy.org/protscale/, accessed on 1 July using Expasy ProtScale tool (https://web.expasy.org/protscale/, accessed on 1 July 2021), 2021), and disor-and der prediction plots according to DISOPRED3 (red, continuous line) and IUPRED2 (red, dotted line). disorder prediction plots according to DISOPRED3 (red, continuous line) and IUPRED2 (red, dotted The separation between the three domains, NTD, MBD, and ID, are indicated by the vertical dashed line). lines.The separation (Right) One of between theconformational the possible three domains,states NTD,ofMBD, and ID, are NTD-MBD-ID, indicated showing theby the two vertical disor- dashed lines. (Right) One of the possible conformational states of dered domains, NTD (yellow) and ID (pink), flanking the folded domain (MBD).NTD-MBD-ID, showing the two disordered domains, NTD (yellow) and ID (pink), flanking the folded domain (MBD). The influence of NTD and ID on MBD may be twofold: (1) unspecific, through steric The influence constraining of NTD and and long-range ID on MBD electrostatic may be twofold: interactions, (1) unspecific, and (2) specific, through through direct in-steric constraining and long-range electrostatic interactions, and teraction with certain regions in the MBD. Both phenomena would result in spatial re-(2) specific, through direct interaction with striction of the MBD certain regions instabilization, and additional the MBD. Both phenomena hindering its abilitywould result to unfold in spatial at moder- restriction of the and ate temperatures MBD and additional denaturant stabilization, concentrations. hindering its ability The interdependence between to the unfold dif- at moderate temperatures ferent domains in MeCP2and wasdenaturant concentrations. observed before. Acrylamide The interdependence collisional quenching, which between the different reports domains the solvent in MeCP2ofwas accessibility the observed before. Acrylamide single tryptophan in MeCP2 (locatedcollisional quenching, in MBD), re- which vealedreports the solvent the structural accessibility coupling betweenofMBD the single and alltryptophan other domains in MeCP2 (located [36]. The in MBD), structure of the MBDthe revealed was influenced structural other domains coupling betweenleading MBD and to the all shielding other domains of W104 from [36]. Thesolvent structure ofexposure, the MBDwithwasall domains contributing, influenced other domains especially leadingNTD,to theID, and TRD. shielding of W104 from solvent If thewith exposure, influence of the disordered all domains contributing, domains on MBD especially NTD,were ID,mostly and TRD. due to electrostatic interactions, If the influence of the disordered domains on MBD were mostlystability the ionic strength would strongly modulate the structural of MBD. due to electrostatic interactions, the ionic strength would strongly modulate the structural stability ofthe It was reported that increasing the intrinsic ionic strength (NaCl 150 mM) decreases MBD. Itstabilization was reported effect thatofincreasing the disordered flanking the intrinsic domains ionic strength(ΔT(NaCl m = 7.8150 °C mM) with decreases zero ionic the strength compared stabilization effect to of ΔT them =disordered 3.4 °C withflanking NaCl 150domains mM). However, (∆Tm =there 7.8 ◦isCstill with a substan- zero ionic strength compared to ∆Tm = 3.4 C with NaCl 150 mM). However, there is still asuggests tial stabilization effect even though ◦ electrostatic interactions are screened. This substantial the steric/conformational stabilization effect even though effects are importantinteractions electrostatic for the stabilization effect on are screened. MBD. This suggests the Regarding the experiments with dsDNA, the same phenomena steric/conformational effects are important for the stabilization effect on MBD. previously observed by fluorescence Regarding the thermal denaturations experiments were alsothe with dsDNA, observed by chemical denaturations: same phenomena previously observed the stabilization effect induced by dsDNA, the larger stabilization effect by fluorescence thermal denaturations were also observed by chemical denaturations: the of dsDNA on NTD- MBD-ID compared stabilization to that on effect induced byMBD, dsDNA, andthethelarger largerstabilization stabilization effect exertedof by dsDNAmethylated on NTD- MBD-ID compared to that on MBD, and the larger stabilization exerted by methylated mCpG-dsDNA compared to unmethylated CpG-dsDNA [34]. Still, there is no clue about the underlying mechanism by which such a moderate difference in the in vitro affinity of MeCP2 for dsDNA between the two methylation states (less than five-fold difference in affinity [34]) is further translated to a considerable difference in stabilization upon binding. This hierarchy we have seen in the unfolding experiments is more coherent with the in vivo situation, where MeCP2 acts as a consistent reader of methylation in chromatin. 5. Conclusions In multidomain proteins, there must be a reciprocal influence of the different consti- tutive domains, an effect that may be qualitatively and quantitatively different in IDPs because of the larger dynamic sampling of structurally diverse conformational states. Con- siderable flexibility and a highly dynamic conformational landscape, large susceptibility to environmental conditions, and potential interactions with partners might cause this interdependence to be conditional and dependent on many intervening factors, as well as provide an additional regulation level for protein conformation and function (i.e., allosteric control). MeCP2 is a multidomain IDP in which MBD, the main domain responsible for Biomolecules 2021, 11, 1216 16 of 18 DNA recognition, is substantially influenced by its flanking disordered domains. Not only does NTD-MBD-ID differ functionally from MBD, but NTD-MBD-ID also shows differ- ential structural features compared to MBD: (1) conformational landscape with higher complexity, where partially unfolded states may be (functionally) relevant; (2) lower unfold- ing cooperativity due to the coexistence of partially unfolded intermediate states; and (3) lower relevant stability than MBD but higher overall stability at moderate temperature. In addition, MBD mutations associated with Rett syndrome have different impacts depending on the molecular context, the isolated MBD or the NTD-MBD-ID construct [55]. The ap- plication of other experimental techniques (e.g., nuclear magnetic resonance, small-angle X-ray scattering) and computational studies applying molecular dynamic simulations with MBD and NTD-MBD-ID remain very challenging considering the disordered nature of the protein, which might reveal additional effects on MBD from NTD and ID. Moreover, it is very likely that the complexity of the behavior of MeCP2 will be higher for the full-length protein containing the six structural domains. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/biom11081216/s1, Figure S1: Circular dichroism, Figure S2: DLS size histograms. Author Contributions: Conceptualization, M.E., O.A. and A.V.-C.; methodology, O.A. and A.V.-C.; software, O.A. and A.V.-C.; validation, D.O.-A., R.C.-G., S.V., O.C.J.-T., M.E., O.A. and A.V.-C.; formal analysis, D.O.-A., R.C.-G., S.V., O.C.J.-T., M.E., O.A. and A.V.-C.; investigation, D.O.-A., R.C.-G., S.V. and O.C.J.-T.; resources, M.E., O.A. and A.V.-C.; data curation, D.O.-A. and R.C.-G.; writing—original draft preparation, D.O.-A., O.A. and A.V.-C.; writing—review and editing, D.O.-A., R.C.-G., S.V., O.C.J.-T., M.E., O.A. and A.V.-C.; visualization, D.O.-A., R.C.-G., S.V., O.C.J.-T., M.E., O.A. and A.V.-C.; supervision, M.E., O.A. and A.V.-C.; project administration, O.A. and A.V.-C.; funding acquisition, O.A. and A.V.-C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Spanish Ministry of Economy and Competitiveness and Euro- pean ERDF Funds (MCIU/AEI/FEDER, EU) (BFU2016-78232-P to A.V.C.; BES-2017-080739 to D.O.A.); Miguel Servet Program from Instituto de Salud Carlos III (CPII13/00017 to O.A.); Fondo de Investi- gaciones Sanitarias from Instituto de Salud Carlos III and European Union (ERDF/ESF, “Investing in your future”) (PI15/00663 and PI18/00349 to O.A.); Diputación General de Aragón (Protein Targets and Bioactive Compounds Group E45_20R to A.V.C. and Digestive Pathology Group B25_20R to O.A.); and the Centro de Investigación Biomédica en Red en Enfermedades Hepáticas y Digestivas (CIBERehd). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available upon reasonable request from the corresponding author. Acknowledgments: The authors acknowledge the help of J.J. Galano-Frutos and J. 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