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The p53 core domain is a molten globule at low pH: functional implications of a partially unfolded structure.

Bom AP, Freitas MS, Moreira FS, Ferraz D, Sanches D, Gomes AM, Valente AP, Cordeiro Y, Silva JL - J. Biol. Chem. (2009)

Bottom Line: This behavior is accompanied by a lack of cooperativity under urea denaturation and decreased stability under pressure when p53C is in acidic pH.Together, these results indicate that p53C acquires a partially unfolded conformation (molten-globule state) at low pH (5.0).The hydrodynamic properties of this conformation are intermediate between the native and denatured conformation. (1)H-(15)N HSQC NMR spectroscopy confirms that the protein has a typical molten-globule structure at acidic pH when compared with pH 7.2.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil.

ABSTRACT
p53 is a transcription factor that maintains genome integrity, and its function is lost in 50% of human cancers. The majority of p53 mutations are clustered within the core domain. Here, we investigate the effects of low pH on the structure of the wild-type (wt) p53 core domain (p53C) and the R248Q mutant. At low pH, the tryptophan residue is partially exposed to the solvent, suggesting a fluctuating tertiary structure. On the other hand, the secondary structure increases, as determined by circular dichroism. Binding of the probe bis-ANS (bis-8-anilinonaphthalene-1-sulfonate) indicates that there is an increase in the exposure of hydrophobic pockets for both wt and mutant p53C at low pH. This behavior is accompanied by a lack of cooperativity under urea denaturation and decreased stability under pressure when p53C is in acidic pH. Together, these results indicate that p53C acquires a partially unfolded conformation (molten-globule state) at low pH (5.0). The hydrodynamic properties of this conformation are intermediate between the native and denatured conformation. (1)H-(15)N HSQC NMR spectroscopy confirms that the protein has a typical molten-globule structure at acidic pH when compared with pH 7.2. Human breast cells in culture (MCF-7) transfected with p53-GFP revealed localization of p53 in acidic vesicles, suggesting that the low pH conformation is present in the cell. Low pH stress also tends to favor high levels of p53 in the cells. Taken together, all of these data suggest that p53 may play physiological or pathological roles in acidic microenvironments.

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Chemically and physically induced unfolding of wt and R248Q p53C. Unfolding was monitored by intrinsic fluorescence and analyzed as the fraction (α) of denaturation for (A) chemical-induced unfolding or (B) CM values for pressure-induced unfolding. Wild-type p53C at pH 7.2 (diamonds) or pH 5.0 (circles) and R248Q at pH 7.2 (triangles) or pH 5.0 (squares) were incubated in: (A) the presence of different urea concentrations (0.5–8.0 m) or (B) compression up to 2.9 kbar. All measurements were performed at 25 °C with 5 μm protein. In B, open symbols correspond to CM values after the return to atmospheric pressure. To analyze the extent of denaturation (α), we used the following equation: α = <ν> − <νi>/<νf> − <νi>, where <ν> is the center of mass at each urea concentration, <νf> is the center of mass in 8 m urea, and <νi> is the center of mass in the absence of urea.
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Figure 6: Chemically and physically induced unfolding of wt and R248Q p53C. Unfolding was monitored by intrinsic fluorescence and analyzed as the fraction (α) of denaturation for (A) chemical-induced unfolding or (B) CM values for pressure-induced unfolding. Wild-type p53C at pH 7.2 (diamonds) or pH 5.0 (circles) and R248Q at pH 7.2 (triangles) or pH 5.0 (squares) were incubated in: (A) the presence of different urea concentrations (0.5–8.0 m) or (B) compression up to 2.9 kbar. All measurements were performed at 25 °C with 5 μm protein. In B, open symbols correspond to CM values after the return to atmospheric pressure. To analyze the extent of denaturation (α), we used the following equation: α = <ν> − <νi>/<νf> − <νi>, where <ν> is the center of mass at each urea concentration, <νf> is the center of mass in 8 m urea, and <νi> is the center of mass in the absence of urea.

Mentions: To analyze differences in the folding cooperativity and conformational stability of the molten globule and native p53C states, we studied the stability of the protein at different pH values against chemical (urea) and physical (pressure) treatments. Chemically induced unfolding of wt or R248Q p53C at pH 7.2 indicated that both proteins unfold to final CM values around 28,700 cm−1. This value is compatible with the complete exposure of aromatic residues to the aqueous environment, suggesting that urea is able to induce complete unfolding. The unfolding of wt and R248Q p53C at pH 7.2 showed a cooperative, two-state denaturation process. The denaturation isotherms of wt and R248Q p53C at pH 5.0 occurred at lower urea concentrations and presented a lack of cooperativity (Fig. 6A) that is typical of a MG conformation.


The p53 core domain is a molten globule at low pH: functional implications of a partially unfolded structure.

Bom AP, Freitas MS, Moreira FS, Ferraz D, Sanches D, Gomes AM, Valente AP, Cordeiro Y, Silva JL - J. Biol. Chem. (2009)

Chemically and physically induced unfolding of wt and R248Q p53C. Unfolding was monitored by intrinsic fluorescence and analyzed as the fraction (α) of denaturation for (A) chemical-induced unfolding or (B) CM values for pressure-induced unfolding. Wild-type p53C at pH 7.2 (diamonds) or pH 5.0 (circles) and R248Q at pH 7.2 (triangles) or pH 5.0 (squares) were incubated in: (A) the presence of different urea concentrations (0.5–8.0 m) or (B) compression up to 2.9 kbar. All measurements were performed at 25 °C with 5 μm protein. In B, open symbols correspond to CM values after the return to atmospheric pressure. To analyze the extent of denaturation (α), we used the following equation: α = <ν> − <νi>/<νf> − <νi>, where <ν> is the center of mass at each urea concentration, <νf> is the center of mass in 8 m urea, and <νi> is the center of mass in the absence of urea.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2807339&req=5

Figure 6: Chemically and physically induced unfolding of wt and R248Q p53C. Unfolding was monitored by intrinsic fluorescence and analyzed as the fraction (α) of denaturation for (A) chemical-induced unfolding or (B) CM values for pressure-induced unfolding. Wild-type p53C at pH 7.2 (diamonds) or pH 5.0 (circles) and R248Q at pH 7.2 (triangles) or pH 5.0 (squares) were incubated in: (A) the presence of different urea concentrations (0.5–8.0 m) or (B) compression up to 2.9 kbar. All measurements were performed at 25 °C with 5 μm protein. In B, open symbols correspond to CM values after the return to atmospheric pressure. To analyze the extent of denaturation (α), we used the following equation: α = <ν> − <νi>/<νf> − <νi>, where <ν> is the center of mass at each urea concentration, <νf> is the center of mass in 8 m urea, and <νi> is the center of mass in the absence of urea.
Mentions: To analyze differences in the folding cooperativity and conformational stability of the molten globule and native p53C states, we studied the stability of the protein at different pH values against chemical (urea) and physical (pressure) treatments. Chemically induced unfolding of wt or R248Q p53C at pH 7.2 indicated that both proteins unfold to final CM values around 28,700 cm−1. This value is compatible with the complete exposure of aromatic residues to the aqueous environment, suggesting that urea is able to induce complete unfolding. The unfolding of wt and R248Q p53C at pH 7.2 showed a cooperative, two-state denaturation process. The denaturation isotherms of wt and R248Q p53C at pH 5.0 occurred at lower urea concentrations and presented a lack of cooperativity (Fig. 6A) that is typical of a MG conformation.

Bottom Line: This behavior is accompanied by a lack of cooperativity under urea denaturation and decreased stability under pressure when p53C is in acidic pH.Together, these results indicate that p53C acquires a partially unfolded conformation (molten-globule state) at low pH (5.0).The hydrodynamic properties of this conformation are intermediate between the native and denatured conformation. (1)H-(15)N HSQC NMR spectroscopy confirms that the protein has a typical molten-globule structure at acidic pH when compared with pH 7.2.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Ressonância Magnética Nuclear de Macromoléculas, Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ 21941-590, Brazil.

ABSTRACT
p53 is a transcription factor that maintains genome integrity, and its function is lost in 50% of human cancers. The majority of p53 mutations are clustered within the core domain. Here, we investigate the effects of low pH on the structure of the wild-type (wt) p53 core domain (p53C) and the R248Q mutant. At low pH, the tryptophan residue is partially exposed to the solvent, suggesting a fluctuating tertiary structure. On the other hand, the secondary structure increases, as determined by circular dichroism. Binding of the probe bis-ANS (bis-8-anilinonaphthalene-1-sulfonate) indicates that there is an increase in the exposure of hydrophobic pockets for both wt and mutant p53C at low pH. This behavior is accompanied by a lack of cooperativity under urea denaturation and decreased stability under pressure when p53C is in acidic pH. Together, these results indicate that p53C acquires a partially unfolded conformation (molten-globule state) at low pH (5.0). The hydrodynamic properties of this conformation are intermediate between the native and denatured conformation. (1)H-(15)N HSQC NMR spectroscopy confirms that the protein has a typical molten-globule structure at acidic pH when compared with pH 7.2. Human breast cells in culture (MCF-7) transfected with p53-GFP revealed localization of p53 in acidic vesicles, suggesting that the low pH conformation is present in the cell. Low pH stress also tends to favor high levels of p53 in the cells. Taken together, all of these data suggest that p53 may play physiological or pathological roles in acidic microenvironments.

Show MeSH
Related in: MedlinePlus