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Energetic landscape of α-lytic protease optimizes longevity through kinetic stability


During the evolution of proteins the pressure to optimize biological activity is moderated by a need for efficient folding. For most proteins, this is accomplished through spontaneous folding to a thermodynamically stable and active native state. However, in the extracellular bacterial α-lytic protease (αLP) these two processes have become decoupled. The native state of αLP is thermodynamically unstable, and when denatured, requires millennia (t1/2 1,800 years)1 to refold. Folding is made possible by an attached folding catalyst, the pro-region, which is degraded on completion of folding, leaving αLP trapped in its native state by a large kinetic unfolding barrier (t1/2 1.2 years)1. αLP faces two very different folding landscapes: one in the presence of the pro-region controlling folding, and one in its absence restricting unfolding. Here we demonstrate that this separation of folding and unfolding pathways has removed constraints placed on the folding of thermodynamically stable proteins, and allowed the evolution of a native state having markedly reduced dynamic fluctuations. This, in turn, has led to a significant extension of the functional lifetime of αLP by the optimal suppression of proteolytic sensitivity.

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Figure 1: Folding free-energy diagrams for thermodynamic compared with kinetic stability. ΔG, free energy difference.
Figure 2: Hydrogen-exchange protection of αLP.
Figure 3: Survival assay at pH 7, 25 °C.
Figure 4: Autolysis compared with global unfolding rate.


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We thank Y. Shibata for assistance with hydrogen exchange experiments, and J. Harris, T. Baird and C. Craik for assistance with trypsin purification. S.S.J. was supported by a Howard Hughes Medical Institute predoctoral fellowship. Research funding was provided by Howard Hughes Medical Institute.

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Jaswal, S., Sohl, J., Davis, J. et al. Energetic landscape of α-lytic protease optimizes longevity through kinetic stability. Nature 415, 343–346 (2002).

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