Electrostatic Effects on the Structure of Intrinsically Disordered Proteins
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Highly flexible and dynamic proteins with negligible tertiary structure are known as intrinsically disordered proteins (IDPs). Current literature suggests the hydrodynamic dimensions of IDPs are dominated by electrostatic effects. However, other studies indicate polyproline II (PPII) content, a non-classical secondary structure, also greatly impacts the structure of IDPs. To test these suggestions in conjunction, we used the disordered N-terminal region (residues 1-93) of the human tumor suppressor protein p53 as our model and hydrodynamic radius (Rh) as a size reporter. We used size exclusion chromatography to measure the Rh and circular dichroism to measure PPII content at a pH range of 4.5-7.0. Previous research indicates proline and alanine as two of the residues with the highest PPII propensities. Hence, we repeated these measurements with two mutants: one in which all proline residues were replaced with glycines (Pro-) and one with all alanines replaced with glycines (Ala-). All three polypeptides have a net charge of -14.99 at pH 7.0 and should be significantly compacted at lower pHs due to a decrease in charge repulsion. As pH was decreased from 7.0 to 4.5, the Rh of the wildtype decreased by 15%, Ala- by 14%, and Pro- by 27%. These results show that prolines increase the spacing between charge groups, weakening electrostatic repulsion. Thus, charge effects are important to IDP structure, but are heavily modulated by PPII content.