Abstract
Intramolecular electrostatic attraction and repulsion strongly influence the conformational sampling of intrinsically disordered proteins and domains (IDPs). In order to better understand this complex relationship, we have used nuclear magnetic resonance to measure side chain pK(a) values and pH-dependent translational diffusion coefficients for the unstructured and highly acidic carboxyl-terminus of γ-tubulin (γ-CT), providing insight into how the net charge of an IDP relates to overall expansion or collapse of the conformational ensemble. Many of the pK(a) values in the γ-CT are shifted upward by 0.3-0.4 units and exhibit negatively cooperative ionization pH profiles, likely due to the large net negative charge that accumulates on the molecule as the pH is raised. pK(a) shifts of this magnitude correspond to electrostatic interaction energies between the affected residues and the rest of the charged molecule that are each on the order of 1 kcal mol(-1) . Diffusion of the γ-CT slowed with increasing net charge, indicative of an expanding hydrodynamic radius (r(H) ). The degree of expansion agreed quantitatively with what has been seen from comparisons of IDPs with different charge content, yielding the general trend that every 0.1 increase in relative charge (|Q|/res) produces a roughly 5% increase in r(H) . While γ-CT pH titration data followed this trend nearly perfectly, there were substantially larger deviations for the database of different IDP sequences. This suggests that other aspects of an IDP's primary amino acid sequence beyond net charge influence the sensitivity of r(H) to electrostatic interactions.