Denatured state ensembles with the same radii of gyration can form significantly different long-range contacts.

Defining the structural, dynamic, and energetic properties of the unfolded state of proteins is critical for an in-depth understanding of protein folding, protein thermodynamics, and protein aggregation. Here we analyze long-range contacts and compactness in two apparently fully unfolded ensembles of the same protein: the acid unfolded state of the C-terminal domain of ribosomal protein L9 in the absence of high concentrations of urea as well as the urea unfolded state at low pH. Small angle X-ray scattering reveals that the two states are expanded with values of Rg differing by <7%. Paramagnetic relaxation enhancement (PRE) nuclear magnetic resonance studies, however, reveal that the acid unfolded state samples conformations that facilitate contacts between residues that are distant in sequence while the urea unfolded state ensemble does not. The experimental PRE profiles for the acid unfolded state differ significantly from these predicted using an excluded volume limit ensemble, but these long-range contacts are largely eliminated by the addition of 8 M urea. The work shows that expanded unfolded states can sample very different distributions of long-range contacts yet still have similar radii of gyration. The implications for protein folding and for the characterization of unfolded states are discussed.