Andrei T. Alexandrescu

Hydrodynamic radii ( R h ‐values) calculated from diffusion coefficients measured by pulse‐field‐gradient nuclear magnetic resonance are compared for folded and unfolded proteins. For native globular proteins, the R h ‐values increase as a power of 0.35 with molecular size, close to the scaling factor of 0.33 predicted from polymer theory. Unfolded proteins were studied under four sets of conditions: in the absence of denaturants, in the presence of 6 M urea, in 95% dimethyl sulfoxide (DMSO), and in 40% hexafluoroisopropanol (HFIP). Scaling factors under all four unfolding conditions are similar (0.49–0.53) approaching the theoretical value of 0.60 for a fully unfolded random coil. Persistence lengths are also similar, except smaller in 95% DMSO, suggesting that the polypeptides are more disordered on a local scale with this solvent. Three of the proteins in our unfolded set have an asymmetric sequence‐distribution of charged residues. While these proteins behave normally in water and 6 M urea, they give atypically low R h ‐values in 40% HFIP and 95% DMSO suggesting they are forming electrostatic hairpins, favored by their asymmetric sequence charge distribution and the low dielectric constants of DMSO and HFIP. While diffusion‐ordered NMR spectroscopy can separate small molecules, we show a number of factors combine to make protein‐sized molecules much more difficult to resolve in mixtures. Finally, we look at the temperature dependence of apparent diffusion coefficients. Small molecules show a linear temperature response, while large proteins show abnormally large apparent diffusion coefficients at high temperatures due to convection, suggesting diffusion reference standards are only useful near 25°C.