Compared to folded proteins, the sequences of intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids. Glutamate is one of the most enriched amino acids in IDPs, while the chemically similar amino acid aspartate is less enriched. So far, the underlying functional differences between glutamates and aspartates in IDPs remain poorly understood. In this study, we examine the differential effects of aspartate and glutamates in IDPs by comparing the function and conformational ensemble of glutamate and aspartate variants of the disordered protein Dss1, using a range of assays, including interaction studies, nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and molecular dynamics simulation. First, we analyze the sequences of the rapidly growing database of experimentally verified IDPs (DisProt) and show that glutamate enrichment is not caused by a taxonomy bias in IDPs. From analyses of local and global structural properties as well as cell growth and protein-protein interactions using a model acidic IDP from yeast and three Glu/Asp variants, we find that while the Glu/Asp variants support similar function and global dimensions, the variants differ in their binding affinities and population of local transient structural elements. We speculate that these local structural differences may play roles in functional diversity, where glutamates can support increased helicity, important for folding and binding, while aspartates support extended structures and form helical caps, as well as playing more relevant roles in, e.g., transactivation domains and ion-binding.

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Measuring the compaction of a protein or complex is key to understand the interactions within and between biomolecules. Experimentally, protein compaction is often probed either by estimating the radius of gyration (Rg) obtained from small-angle X-ray scattering (SAXS) experiments or the hydrodynamic radius (Rh) obtained for example by pulsed field gradient nuclear magnetic resonance (PFG NMR) spectroscopy. PFG NMR experiments generally report on the translational diffusion coefficient, which in turn can be used to estimate Rh using an internal standard. Here, we examine the use of 1,4-dioxane as an internal NMR standard to account for sample viscosity and uncertainty about the gradient strength. Specifically, we revisit the basis for the commonly used reference value for the Rh of dioxane (2.12 Å) that is used to convert measured diffusion coefficients into a hydrodynamic radius. We follow the same approach that was used to establish the current reference value for the Rh by measuring SAXS and PFG NMR data for a set of seven different proteins and using these as standards. Our analysis shows that the current Rh reference value for 1,4-dioxane Rh (2.12 Å) is underestimated, and we instead suggest a new value of 2.27 Å ± 0.04 Å. Using this updated reference value results in a ≈7% increase in Rh values for proteins whose hydrodynamic radius have been measured by PFG NMR. We discuss the implications for ensemble descriptions of intrinsically disordered proteins and evaluation of effect resulting from for example ligand binding, post-translational modifications, or changes to the environment.

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