The isolated armadillo-repeat domain of Plakophilin 1 is a monomer in solution with a low conformational stability

Giudici, Ana Marcela; Hernández-Cifre, José G.; Cámara-Artigas, A.; Hornos, Felipe; Martínez‐Rodríguez, Sergio; Álvarez-Pérez, Juan Carlos; Díaz-Cano, Inés; Fárez-Vidal, María Esther; Neira, José L.

doi:10.1016/j.jsb.2020.107569

Cited by 8 publications

(8 citation statements)

References 40 publications

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“…The first conclusion of our work is that N-MDM2 in solution had a rather low stability (with an apparent thermal denaturation midpoint of ~55 • C) in a narrow pH interval (between pH 7.0 and 10.0). A similar low stability has been reported in other prevalently αhelical proteins [31][32][33][34], where it has been associated with a large flexibility. It is interesting to compare our results with those obtained with model proteins such as chicken and equine lysozyme [35,36].…”

Section: Discussionsupporting

confidence: 78%

“…The pH of each sample was measured after completion of pH denaturations with an ultra-thin Aldrich electrode in a Radiometer (Copenhagen) pH meter. The salts and acids used in the buffers have been described elsewhere [34]. The wavelength-averaged emission intensity (also called the spectrum mass center), <λ>, was calculated as described [34].…”

Section: Intrinsic Fluorescencementioning

confidence: 99%

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Conformational Stability of the N-Terminal Region of MDM2

Rizzuti,

Abian,

Velazquez-Campoy

et al. 2023

Molecules

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MDM2 is an E3 ubiquitin ligase which is crucial for the degradation and inhibition of the key tumor-suppressor protein p53. In this work, we explored the stability and the conformational features of the N-terminal region of MDM2 (N-MDM2), through which it binds to the p53 protein as well as other protein partners. The isolated domain possessed a native-like conformational stability in a narrow pH range (7.0 to 10.0), as shown by intrinsic and 8-anilinonapthalene-1-sulfonic acid (ANS) fluorescence, far-UV circular dichroism (CD), and size exclusion chromatography (SEC). Guanidinium chloride (GdmCl) denaturation followed by intrinsic and ANS fluorescence, far-UV CD and SEC at physiological pH, and differential scanning calorimetry (DSC) and thermo-fluorescence experiments showed that (i) the conformational stability of isolated N-MDM2 was very low; and (ii) unfolding occurred through the presence of several intermediates. The presence of a hierarchy in the unfolding intermediates was also evidenced through DSC and by simulating the unfolding process with the help of computational techniques based on constraint network analysis (CNA). We propose that the low stability of this protein is related to its inherent flexibility and its ability to interact with several molecular partners through different routes.

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Section: Discussionsupporting

confidence: 78%

Section: Intrinsic Fluorescencementioning

confidence: 99%

Conformational Stability of the N-Terminal Region of MDM2

Rizzuti,

Abian,

Velazquez-Campoy

et al. 2023

Molecules

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“…It was observed that, at pH 7.0 and 8.0, the thermal denaturation midpoints obtained by intrinsic fluorescence were slightly higher than those measured by CD. We have reported similar behavior for other large monomeric proteins which unfold irreversibly, by following the changes with the same two spectroscopic probes [42,43]. Such behavior has been explained as being due to the large irreversibility of the process, which is probably increased at the high basic pH values where thermal denaturations occurred.…”

Section: The Ph Conformational Changes Of Bshprk/p In Solutionsupporting

confidence: 59%

The Histidine Phosphocarrier Kinase/Phosphorylase from Bacillus Subtilis Is an Oligomer in Solution with a High Thermal Stability

Neira

Cámara-Artigas

Hernández-Cifre

et al. 2021

IJMS

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The histidine phosphocarrier protein (HPr) kinase/phosphorylase (HPrK/P) modulates the phosphorylation state of the HPr protein, and it is involved in the use of carbon sources by Gram-positive bacteria. Its X-ray structure, as concluded from crystals of proteins from several species, is a hexamer; however, there are no studies about its conformational stability, and how its structure is modified by the pH. We have embarked on the conformational characterization of HPrK/P of Bacillus subtilis (bsHPrK/P) in solution by using several spectroscopic (namely, fluorescence and circular dichroism (CD)) and biophysical techniques (namely, small-angle X-ray-scattering (SAXS) and dynamic light-scattering (DLS)). bsHPrK/P was mainly a hexamer in solution at pH 7.0, in the presence of phosphate. The protein had a high conformational stability, with an apparent thermal denaturation midpoint of ~70 °C, at pH 7.0, as monitored by fluorescence and CD. The protein was very pH-sensitive, precipitated between pH 3.5 and 6.5; below pH 3.5, it had a molten-globule-like conformation; and it acquired a native-like structure in a narrow pH range (between pH 7.0 and 8.0). Guanidinium hydrochloride (GdmCl) denaturation occurred through an oligomeric intermediate. On the other hand, urea denaturation occurred as a single transition, in the range of concentrations between 1.8 and 18 µM, as detected by far-UV CD and fluorescence.

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“…RYBP and ARM-PKP1 were purified as previously described [25,51]. Protein concentrations were determined through UV absorbance, employing an extinction coefficient at 280 nm estimated from the numbers of tyrosines and tryptophans in each protein [56].…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…The structure of the ARM-repeat domain of PKP1 (ARM-PKP1) has been solved via X-ray (Figure 1B); it contains nine ARM motifs and includes a large basic patch serving as a binding site region for most of its partners [48][49][50]. We have previously shown that isolated ARM-PKP1 is a monomer in solution, and it has a low conformational stability [51]. Furthermore, it interacts with the sterile alpha motif (SAM) of p73 [52] and with NUPR1, an IDP intervening in the development of pancreatic cancer [53].…”

Section: Introductionmentioning

confidence: 99%

Unveiling the Binding between the Armadillo-Repeat Domain of Plakophilin 1 and the Intrinsically Disordered Transcriptional Repressor RYBP

Araujo-Abad,

Rizzuti,

Vidal

et al. 2024

Biomolecules

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Plakophilin 1 (PKP1), a member of the p120ctn subfamily of the armadillo (ARM)-repeat-containing proteins, is an important structural component of cell–cell adhesion scaffolds although it can also be ubiquitously found in the cytoplasm and the nucleus. RYBP (RING 1A and YY1 binding protein) is a multifunctional intrinsically disordered protein (IDP) best described as a transcriptional regulator. Both proteins are involved in the development and metastasis of several types of tumors. We studied the binding of the armadillo domain of PKP1 (ARM-PKP1) with RYBP by using in cellulo methods, namely immunofluorescence (IF) and proximity ligation assay (PLA), and in vitro biophysical techniques, namely fluorescence, far-ultraviolet (far-UV) circular dichroism (CD), and isothermal titration calorimetry (ITC). We also characterized the binding of the two proteins by using in silico experiments. Our results showed that there was binding in tumor and non-tumoral cell lines. Binding in vitro between the two proteins was also monitored and found to occur with a dissociation constant in the low micromolar range (~10 μM). Finally, in silico experiments provided additional information on the possible structure of the binding complex, especially on the binding ARM-PKP1 hot-spot. Our findings suggest that RYBP might be a rescuer of the high expression of PKP1 in tumors, where it could decrease the epithelial–mesenchymal transition in some cancer cells.

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The isolated armadillo-repeat domain of Plakophilin 1 is a monomer in solution with a low conformational stability

Cited by 8 publications

References 40 publications

Conformational Stability of the N-Terminal Region of MDM2

Conformational Stability of the N-Terminal Region of MDM2

The Histidine Phosphocarrier Kinase/Phosphorylase from Bacillus Subtilis Is an Oligomer in Solution with a High Thermal Stability

Unveiling the Binding between the Armadillo-Repeat Domain of Plakophilin 1 and the Intrinsically Disordered Transcriptional Repressor RYBP

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