Approximately 30% of the proteins in the human proteome are intrinsically disordered (IDPs) or contain long disordered regions (IDRs). IDPs play a central role in key cellular regulatory pathways and are implicated in devastating diseases such as cancer, diabetes, cardiovascular disease, and neurodegenerative disease. Disordered proteins are highly flexible and undergo transient and dynamic intramolecular and intermolecular interactions that are central to their regulatory functions. Molecular level characterization of the numerous human regulatory proteins that contain both structured and disordered domains represents an enormous challenge to the traditional methods of structural biology. Most studies to date have relied upon a reductionist approach, in which the ordered and disordered regions are investigated in isolation. However, within the cell, the different domains of a given protein act synergistically to allow it to perform its biological function and a full understanding of the underlying molecular mechanism can only be achieved through a holistic, rather than reductionist, approach. The overarching goal of this proposal is to utilize a non-reductionist approach to characterize the structural ensemble and dynamics of the full-length tumor suppressor p53, which contains both globular and disordered domains. An innovative, intein-based segmental isotope labeling strategy and advanced NMR methods will be utilized to characterize the conformational ensemble and dynamics of full-length p53, both free and in its complexes with specific and non-specific DNA targets. The disordered N- and C- terminal regions of p53 regulate DNA binding through dynamic intramolecular and intermolecular interactions that are modulated by constitutive and stress-induced posttranslational modifications. This research will provide new molecular level insights into the mechanisms by which this important tumor suppressor is dynamically regulated by the cascade of phosphorylation and acetylation that is triggered by genotoxic stress.
The tumor suppressor p53 is of central importance in the prevention of cancer. The proposed research will address the molecular mechanisms by which the integral disordered domains of p53 regulate its transcriptional activity in response to damage to cellular DNA.
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