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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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Size-exclusion HPLC analysis of oligomeric states of native and MG p53C. Normalized fluorescence at 280 nm was detected during gel filtration chromatography of wt p53C at pH 7.2 (solid line), pH 5.0 (dotted line), or incubated with 3 m GdmCl (dashed line).
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Figure 4: Size-exclusion HPLC analysis of oligomeric states of native and MG p53C. Normalized fluorescence at 280 nm was detected during gel filtration chromatography of wt p53C at pH 7.2 (solid line), pH 5.0 (dotted line), or incubated with 3 m GdmCl (dashed line).

Mentions: To investigate the hydrodynamic properties of p53C at pH 7.2 and pH 5.0, we analyzed the proteins by gel filtration chromatography (Fig. 4) and verified that both isoforms have a lower retention time at pH 5.0 than at pH 7.2 (Fig. 4 and data not shown), indicating that there is an increase in the hydrodynamic volume for both proteins at low pH. The data show a retention time of 13 min for the native conformation at pH 7.2 (Fig. 4, solid line), 12 min for the MG conformation (Fig. 4, dotted line), and 10 min for a completely denatured protein (Fig. 4, dashed line). The hydrodynamic radiuses (Rg) derived from the elution volumes and from the calibration of the column with standard proteins were: 1) 17.6 ± 0.5 Å for p53C at pH 7.2; 2) 19.3 ± 0.4 Å for p53C at pH 5.0; and 3) 30.9 ± 0.5 Å for denatured p53C.


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)

Size-exclusion HPLC analysis of oligomeric states of native and MG p53C. Normalized fluorescence at 280 nm was detected during gel filtration chromatography of wt p53C at pH 7.2 (solid line), pH 5.0 (dotted line), or incubated with 3 m GdmCl (dashed line).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Size-exclusion HPLC analysis of oligomeric states of native and MG p53C. Normalized fluorescence at 280 nm was detected during gel filtration chromatography of wt p53C at pH 7.2 (solid line), pH 5.0 (dotted line), or incubated with 3 m GdmCl (dashed line).
Mentions: To investigate the hydrodynamic properties of p53C at pH 7.2 and pH 5.0, we analyzed the proteins by gel filtration chromatography (Fig. 4) and verified that both isoforms have a lower retention time at pH 5.0 than at pH 7.2 (Fig. 4 and data not shown), indicating that there is an increase in the hydrodynamic volume for both proteins at low pH. The data show a retention time of 13 min for the native conformation at pH 7.2 (Fig. 4, solid line), 12 min for the MG conformation (Fig. 4, dotted line), and 10 min for a completely denatured protein (Fig. 4, dashed line). The hydrodynamic radiuses (Rg) derived from the elution volumes and from the calibration of the column with standard proteins were: 1) 17.6 ± 0.5 Å for p53C at pH 7.2; 2) 19.3 ± 0.4 Å for p53C at pH 5.0; and 3) 30.9 ± 0.5 Å for denatured p53C.

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