Protein-DNA binding correlates with structural thermostability for the full-length human p53 protein.

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• The tumor suppressor p53 protein plays a critical role in cellular responses to stress.
• Understanding the structural stability and DNA-binding activity of p53 is essential for its function.

Purpose of the Study:

• To investigate the correlation between the thermal stability of the full-length p53 protein and its functional DNA binding capabilities.
• To assess the DNA binding activity of p53 across a range of temperatures.

Main Methods:

• Purification of full-length p53 protein from Escherichia coli.
• Circular dichroism (CD) spectroscopy to analyze protein structure and thermal stability.
• DNA binding assays to determine binding affinity (K(d)) to dsDNA targets at various temperatures.

Main Results:

• Full-length p53 exhibits remarkable thermal stability, with an unfolding midpoint around 73°C.
• Significant beta-sheet structure, indicative of DNA binding potential, persists even at 100°C.
• The protein retains high-affinity DNA binding (nM range) up to 50°C.
• Reactivity to the Ab1620 antibody is not a reliable indicator of functional DNA binding activity.

Conclusions:

• The DNA binding domain of full-length p53 is stable and functionally active over a broad temperature range.
• p53 can bind consensus dsDNA targets effectively in its latent form, without activators.
• Epitope Ab1620 reactivity does not correlate with the functional DNA binding capacity of p53.