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bioRxiv · 10.1101/2020.08.30.274340

Exploring the evolutionary history of kinetic stability in the alpha-lytic protease family

Nixon, C. F.·Lim, S. A.·Sailer, Z. R.·Zheludev, I. N.·Gee, C. L.·Kelch, B. A.·Harms, M. J.·Marqusee, S.
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Abstract

In addition to encoding the final tertiary fold and stability, the primary sequence of a protein encodes the folding trajectory and kinetic barriers that determines the speed of folding. How these kinetic barriers are encoded by the sequence is not well understood. Here, we use evolutionary sequence variation in the alpha-lytic protease (LP) protein family to probe the relationship between sequence and energy landscape. LP has an unusual energy landscape: the native state of LP is not the most thermodynamically favored conformation and, instead, it remains folded due to a large kinetic barrier preventing unfolding. In order to fold, LP utilizes an N-terminal pro region of similar size to the protease itself that functions as a folding catalyst. Once folded, the pro region is removed, and the native state does not unfold on a biologically relevant timescale. Without the pro region, LP folds on the order of millennia. A phylogenetic search uncovers LP homologs with a wide range of pro-region sizes, including some with no pro region at all. In the resulting phylogenetic tree, these homologs cluster by pro-region size. Homologs naturally lacking pro regions are thermodynamically stable, fold much faster than LP, yet retain the same fold as LP. Key amino acids thought to contribute to LPs extreme kinetic stability are lost in these homologs, further supporting their role in kinetic stability. This study highlights how the entire energy landscape plays an important role in determining the evolutionary pressures on and changes to the protein sequence.

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Nixon, C. F., Lim, S. A., Sailer, Z. R., Zheludev, I. N., Gee, C. L., Kelch, B. A., Harms, M. J., Marqusee, S.. 2020-08-31. Exploring the evolutionary history of kinetic stability in the alpha-lytic protease family. https://doi.org/10.1101/2020.08.30.274340

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