Chain Compaction and Synergistic Destabilization of Globular Proteins by Mixture of Denaturants

Abstract

Characterization of unfolded states is essential to understand the mechanism of protein folding. This study examines the unfolded states of RNase A and α-LA in the mixture of ′′structurally-similar′′ chemical denaturants, arginine (Arg) and guanidinium chloride (Gdm). Thermal- and chemical-unfolding experiments show that the mixture of denaturants synergistically destabilizes the proteins. Volumetric analysis shows that partial molar volume (V°) and adiabatic compressibility (Ks) of the proteins decreases with increasing [Gdm]. This suggests that the proteins might be gradually losing their intra-molecular interactions resulting in decreased internal cavity. The addition of Arg to the Gdm-unfolded states decreases both V° and Ks values indicating compaction of the protein chains. This could be attributed to reduction in the hydration of surface-exposed residues of the proteins upon direct interaction with Arg and increased non-native contacts. The results provide a direct experimental evidence for the compaction of unfolded protein chain upon addition of another denaturant.