Residual Structure in the Denatured State of a Three-Helix Bundle Protein

Abstract

The denatured state of a protein is canonically described as an ensemble of non-interacting random coil conformations. By studying a helical bundle protein under denaturing conditions, we have found that residual structure persists in its denatured state. Our work uses the upstream Ubiquitin-Associated domain, UBA(1), of the HHR23A protein as a model system to study residual structure in the denatured state ensemble. Multi-dimensional nuclear magnetic resonance (NMR) experiments 1H-15N HSQC, HNCO, and HNCA were used to find backbone atom secondary chemical shifts which correspond to protein secondary structure with amino acid resolution. By comparing chemical shifts in moderate- to highly denaturing conditions of 4M, 5M, and 6M guanidine HCl to a reference state of 7M GdnHCl, UBA(1) shows residual helical content among all three helices. In contrast, the helical propensity is predicted by the program Agadir to be higher in helix 2 and negligible elsewhere, with the assumption that helices do not interact. The presence of relatively uniform helical content among all three helices may indicate stabilizing tertiary interactions despite highly denaturing conditions. To further probe tertiary interactions, single-residue mutants of UBA(1) will be used to determine if residues in the hydrophobic core or turn regions are contributing to stability of residual structure in the denatured state.