Reversible aggregation plays a crucial role on the folding landscape of p53 core domain
Daniella Ishimaru 1, Luis M. T. R. Lima 1, Lenize F. Maia 1, Priscila M. Lopez 1, Ana P. Ano Bom 1, Ana P. Valente 1 and Jerson Silva 1*
1 Universidade Federal do Rio de Janeiro
* To whom correspondence should be addressed. E-mail: jerson{at}bioqmed.ufrj.br.
Submitted on April 19, 2004
Revised on May 24, 2004
Accepted on 23 July 2004
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Abstract |
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The role of tumor suppressor protein p53 in cell cycle control depends on its flexible and partially unstructured conformation, which makes it crucial to understand its folding landscape. Here we report an intermediate structure of the core domain of the tumor suppressor protein p53 (p53C) during equilibrium and kinetic folding/unfolding transitions induced by guanidinium chloride (GdmCl). This partially folded structure was undetectable when investigated by intrinsic fluorescence. Indeed, the fluorescence data showed a simple two-state transition. On the other hand, analysis of far ultraviolet circular dichroism in 1.0 M GdmCl demonstrated a high content of secondary structure, and the use of an extrinsic fluorescent probe, bis-ANS, indicated an increase in exposure of the hydrophobic core at 1 M GdmCl. This partially folded conformation of p53C was plagued by aggregation, as suggested by one-dimensional NMR and demonstrated by light scattering and gel-filtration chromatography. Dissociation by high pressure of these aggregates reveals the reversibility of the process and that the aggregates have water-excluded cavities. Kinetic measurements show that the intermediate formed in a parallel reaction between unfolded and folded structures and that it is under fine energetic control. They are not only crucial to the folding pathway of p53C but may explain as well the vulnerability of p53C to undergo departure of the native to an inactive state, which makes the cell susceptible to malignant transformation.
Key Words:
Fluorescence, Folding intermediates, Hydrostatic pressure, Protein aggregation, Protein denaturation, Tumor suppresor protein p53
