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, C Delta 4 (a 4-residue carboxyterminal deletion variant of FynSH3) appears to be largely unfolded even in the absence of denaturant. Nevertheless, C Delta 4 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 C Delta 4. 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. (C) 2009 Elsevier Ltd. All rights reserved.