Exploring the sequence and structural determinants of the energy landscape from thermodynamically stable and...

A protein's energy landscape, all accessible conformations, their populations, and dynamics of interconversion, is encoded in its primary sequence. While how this sequence encodes a protein's native state is well understood, how it encodes the dynamics, such as the kinetic barriers for unfolding and refolding, is not. Here we have looked at two subtiliase homologs from Bacillus subtilis, Intracellular Subtilisin Protease 1 (ISP1) and Subtilisin E (SbtE), that are expected to have very different dynamics. ISP1, an intracellular protein, 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 the mature proteins have been previously characterized; here we 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 ISP1's pro-domain has limited impact on the energy landscape of the mature protein. For SbtE, the protein is thermodynamically unstable and kinetically trapped without the pro-domain. The pro-domains' effects on the flexibility of the core of the proteins are different: in the absence of its pro-domain, ISP1's core becomes more flexible, while SbtE's core becomes more rigid. ISP1 contains a conserved insertion, which points to a potential source for these differences. These homologs show how changes in the primary sequence can dramatically alter a protein's energy landscape and highlight the need for large-scale, high-throughput studies on the relationship between primary sequence and conformational dynamics.