Exploring the sequence and structural determinants of the energy landscape from thermodynamically stable and kinetically trapped subtilisins: ISP1 and SbtE

Abstract

A proteins energy landscape, all accessible conformations, their populations, and dynamics of interconversion, is encoded in its primary sequence. While how a proteins primary sequence encodes its native state is well understood, how sequence encodes the kinetic barriers such as unfolding and refolding is not. Here we have looked at two subtiliase homologs from the Bacillus subtilis, Intracellular Subtilisin Protease 1 (ISP1) and Subtilisin E (SbtE), that are expected to have very different dynamics. As an intracellular protein, ISP1 has a small pro-domain thought to act simply as a zymogen, whereas the extracellular SbtE has a large pro-domain required for folding. The stability and kinetics of ISP1 and ProSbtE have been previously characterized. We now directly compare their energy landscapes with and without the pro-domain, examining global and local energetics of the mature proteases and the effect of each pro-domain. We find that ISP1s pro-domain has limited impact on the energy landscape of the mature protein while without the pro-domain, SbtE is thermodynamically unstable and kinetically trapped. The pro-domains show opposite effects on the flexibility of the core of the protein: in the absence of its pro-domain, ISP1s core becomes more flexible while SbtEs core becomes more rigid. ISP1 contains a unique conserved insertion, which points to a potential source for these differences. These homologs are an example of how changes in the primary sequence can dramatically alter a proteins energy landscape, and highlight the need for large scale, high-throughput studies on the relationship between primary sequence and conformational dynamics.