Non-sequence-specific interactions can account for the compaction of proteins unfolded under "native" conditions.

Proteins unfolded by high concentrations of chemical denaturants adopt expanded, largely structure-free ensembles of conformations that are well approximated as random coils. In contrast, globular proteins unfolded under less denaturing conditions (via mutations, or transiently unfolded after a rapid jump to native conditions) and molten globules (arising due to mutations or cosolvents) are often compact. Here we explore the origins of this compaction using a truncated equilibrium-unfolded variant of the 57-residue FynSH3 domain. As monitored by far-UV circular dichroism, NMR spectroscopy, and hydrogen-exchange kinetics, CDelta4 (a 4-residue carboxy-terminal deletion variant of FynSH3) appears to be largely unfolded even in the absence of denaturant. Nevertheless, CDelta4 is quite compact under these conditions, with a hydrodynamic radius only slightly larger than that of the native protein. In order to understand the origins of this molten-globule-like compaction, we have characterized a random sequence polypeptide of identical amino acid composition to CDelta4. Notably, we find that the hydrodynamic radius of this random sequence polypeptide also approaches that of the native protein. Thus, while native-like interactions may contribute to the formation of compact "unfolded" states, it appears that non-sequence-specific monomer-monomer interactions can also account for the dramatic compaction observed for molten globules and the "physiological" unfolded state.