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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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Secondary structure of the MG state. Wild-type (A) and R248Q p53C (B), at 10 μm, were incubated in solutions with different pH values, and the molar ellipticity at 218 nm was collected. C, circular dichroism spectra of wt p53C at pH 5.0 (dotted line) and pH 7.2 (solid line).
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Figure 3: Secondary structure of the MG state. Wild-type (A) and R248Q p53C (B), at 10 μm, were incubated in solutions with different pH values, and the molar ellipticity at 218 nm was collected. C, circular dichroism spectra of wt p53C at pH 5.0 (dotted line) and pH 7.2 (solid line).

Mentions: Because the MG conformation is characterized by maintenance of or increase in secondary structure (48, 49), we also measured circular dichroism to investigate structural changes due to low pH (Fig. 3). To follow changes in p53C predominantly β-sheet secondary structure, CD ellipticity values were monitored at 218 nm for p53C incubated at various pH values (Fig. 3, A and B). wt and R248Q p53C showed a significant increase in secondary structure at pH 5.0 (Fig. 3), an observation typical of other MG states (14, 19). There is a 1.7-fold gain in β-sheet secondary structure content for wt p53C at pH 5.0 in comparison with the same protein incubated at pH 7.2 as determined by the [θ] value obtained at 212 nm at both conditions.


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)

Secondary structure of the MG state. Wild-type (A) and R248Q p53C (B), at 10 μm, were incubated in solutions with different pH values, and the molar ellipticity at 218 nm was collected. C, circular dichroism spectra of wt p53C at pH 5.0 (dotted line) and pH 7.2 (solid line).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Secondary structure of the MG state. Wild-type (A) and R248Q p53C (B), at 10 μm, were incubated in solutions with different pH values, and the molar ellipticity at 218 nm was collected. C, circular dichroism spectra of wt p53C at pH 5.0 (dotted line) and pH 7.2 (solid line).
Mentions: Because the MG conformation is characterized by maintenance of or increase in secondary structure (48, 49), we also measured circular dichroism to investigate structural changes due to low pH (Fig. 3). To follow changes in p53C predominantly β-sheet secondary structure, CD ellipticity values were monitored at 218 nm for p53C incubated at various pH values (Fig. 3, A and B). wt and R248Q p53C showed a significant increase in secondary structure at pH 5.0 (Fig. 3), an observation typical of other MG states (14, 19). There is a 1.7-fold gain in β-sheet secondary structure content for wt p53C at pH 5.0 in comparison with the same protein incubated at pH 7.2 as determined by the [θ] value obtained at 212 nm at both conditions.

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