Increasing stability reduces conformational heterogeneity in a protein folding intermediate ensemble

Abstract

A multi-site, time-resolved fluorescence resonance energy transfer methodology has been used to study structural heterogeneity in a late folding intermediate ensemble, I_L, of the small protein barstar. Four different intra-molecular distances have been measured within the structural components of I_L. The I_L ensemble is shown to consist of different sub-populations of molecules, in each of which one or more of the four distances are native-like and the remaining distances are unfolded-like. In very stable conditions that favor formation of I_L, all four distances are native-like in most molecules. In less stable conditions, one or more distances are unfolded-like. As stability is decreased, the proportion of molecules with unfolded-like distances increases. Thus, the results show that protein folding intermediates are ensembles of different structural forms, and they demonstrate that conformational entropy increases as structures become less stable. These observations provide direct experimental evidence in support of a basic tenet of energy landscape theory for protein folding, that available conformational space, as represented by structural heterogeneity in I_L, becomes restricted as the stability is increased. The results also vindicate an important prediction of energy landscape theory, that different folding pathways may become dominant under different folding conditions. In more stable folding conditions, uniformly native-like compactness is achieved during folding to I_L, whereas in less stable conditions, uniformly native-like compactness is achieved only later during the folding of I_L to N.