Intrinsically disordered proteins (IDPs) do not form stable tertiary structures like their ordered partners. They exist as heterogeneous ensembles that fluctuate over a time scale. Intrinsically disordered regions and proteins are found across different phyla and exert crucial biological functions. They exhibit transient secondary structures in their free state and become folded upon binding to their protein partners via a mechanism called coupled folding and binding. Some IDPs form alpha helices when bound to their protein partners. We observed a set of cancer associated IDPs where the helical binding segments of IDPs are flanked by prolines on both the sides. Helix-breaking prolines are frequently found in IDPs flanking the binding segment and are evolutionarily conserved across phyla. Two studies have shown that helix flanking prolines modulate the function of IDPs by regulating the levels of disorder in their free state and in turn regulating the binding affinities to their partners. We aimed to study if this is a common phenomenon in IDPs that exhibit similar pattern in the conservation of helix flanking prolines. We chose to test the hypothesis in c-Myb-KIX : IDP-target system in which the disordered protein exhibits high residual helicity levels in its free state.
c-Myb is a hematopoietic regulator that plays a crucial role in cancer by binding to the KIX domain of CBP. Studying the functional regulation of c-Myb by modulating the disorder levels in c-Myb and in IDPs in general provides a better understanding of the way IDPs function and can be used in therapeutic strategies as IDPs are known to be involved in regulating various cellular processes and diseases. To study the effect of conserved helix flanking prolines on the residual helicity levels of c-Myb and its binding affinity to the KIX domain of CBP, we mutated the prolines to alanines. Mutating prolines to alanines increased the helicity levels of c-Myb in its free state. This small increase in the helicity levels of a highly helical c-Myb showed almost no effect on the binding affinity between cMyb and KIX. We hypothesized that there is a helical threshold for coupled folding and binding beyond which helicity levels of the free state IDP have no effect on its binding to their ordered protein partner. To test this hypothesis, we mutated solvent exposed amino acid residues in c-Myb that reduce its overall helicity and studied its effect on the binding affinity between c-Myb and KIX. Over a broad range of reduction in helicity levels of the free state did not show an effect on the binding affinity but beyond a certain level, decrease in helicity levels showed pronounced effects on the binding affinity between c-Myb and KIX.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-8633 |
| Date | 03 November 2017 |
| Creators | Poosapati, Anusha |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
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