Denatured State Ensembles with the Same Radii of Gyration Can Form Significantly Different Long-Range Contacts Bowu Luan, † Nicholas Lyle, ‡ Rohit V. Pappu, ‡ and Daniel P. Raleigh* ,†,§ † Department of Chemistry, Stony Brook University, Stony Brook, New York 11794-3400, United States ‡ Department of Biomedical Engineering, Washington University in St. Louis, One Brookings Drive, Campus Box 1097, St. Louis, Missouri 63130-4899, United States § Graduate Program in Biochemistry and Structural Biology and Graduate Program in Biophysics, Stony Brook University, Stony Brook, New York 11794, United States * S Supporting Information ABSTRACT: Defining the structural, dynamic, and energetic properties of the unfolded state of proteins is critical for an in- depth understanding of protein folding, protein thermody- namics, 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 R g 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. Q uantitative characterization of denatured state ensem- bles (DSEs) of proteins, also termed the unfolded or denatured state, is important for understanding the mechanism of protein folding. The DSE is the starting point of protein folding, the thermodynamic reference state for protein stability, and it can be targeted by rational protein design. 1-7 Studies of DSEs can also reveal factors that impact protein misfolding and modulate the tendency for protein aggregation in vitro and in vivo and amyloid formation. 8-12 The exploration of the mechanisms and biological function of intrinsically disordered proteins (IDPs) largely depends on the characterization of the properties of unstructured and partially structured states and therefore has much in common with studies of the DSE. 13,14 The properties of the DSE can vary considerably depending upon the conditions used to populate it. Under near-native conditions, the DSE can be compact with significant residual structure, while more expanded and less structured DSEs are usually populated under strongly denatured conditions. Small angle X-ray scattering (SAXS) is frequently used to study the overall compactness of the DSEs and provides the radius of gyration (R g ) and in favorable cases more information. 15-20 DSEs that have the same value of R g are often assumed to be similarly unfolded. 16,17,21 Under strongly denaturing conditions, the DSE expands to make favorable interactions with the solvent, and the R g of proteins without disulfide cross-links follows a nontrivial power law relationship, which scales with the number of amino acids in the peptide chain, N, as N 0.59 . 16,17,22 Similar scaling behavior is observed for polymers modeled as self-avoiding random walks. 23 Observation of an R g value consistent with this scaling is often taken to mean a protein is fully unfolded; however, this scaling does not preclude the possibility of detectable, low- likelihood native and non-native contacts within expanded DSEs, even under strongly denaturing conditions. 2-6,8,13,24-36 However, it is unclear if different DSEs generated for the same protein under different conditions, all with similar R g values, will exhibit similar patterns of and likelihoods of native and non-native contacts. This issue is important because such contacts might contribute directly to folding and might influence the tendency to aggregate. It is also important because it potentially highlights the need to go beyond measurements of R g alone as an adjudicator of the degree of unfoldedness and as a descriptor of unfolded states. Here we examine the 92-residue C-terminal domain of ribosomal protein L9 in the acid-induced DSE and in the low- pH urea-induced DSE to determine if the conformational Received: June 27, 2013 Revised: November 15, 2013 Published: November 26, 2013 Article pubs.acs.org/biochemistry © 2013 American Chemical Society 39 dx.doi.org/10.1021/bi4008337 | Biochemistry 2014, 53, 39-47