1. Urea Denaturation, Zinc Binding, and DNA Binding Assays of Mutant p53 DNA-binding Domains and Full-length Proteins
- Author
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Jeung- Ha, Xin Yu, Darren Carpizo, and Stewart Loh
- Subjects
Biology (General) ,QH301-705.5 - Abstract
In the cell, the thermodynamic stability of a protein – and hence its biological activity – can change dramatically as a result of perturbations in its amino acid sequence and the concentration of stabilizing ligands. This interplay is particularly evident in zinc-binding transcription factors such as the p53 tumor suppressor, whose DNA-binding activity can critically depend on levels of intracellular zinc as well as point mutations that alter either metal binding or folding stability. Separate protocols exist for determining a protein’s metal affinity and its folding free energy. These properties, however, are intimately connected, and a technique is needed to integrate these measurements. Our protocols employ common non-fluorescent and fluorescent zinc chelators to control and report on free Zn2+ concentration, respectively, combined with biophysical assays of full-length human p53 and its DNA-binding domain. Fitting the data to equations that contain stability and metal-binding terms results in a more complete picture of how metal-dependent proteins can lose and gain DNA-binding function in a range of physiological conditions.Graphic abstract:Figure 1. Raising intracellular zinc can restore tumor-suppressing function to p53 that has been unfolded by missense mutation or cellular conditions
- Published
- 2021
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