Early Steps in the Unfolding of Thermolysin-like Proteases*

Several series of site-directed mutations in thermolysin-like proteases are presented that show remarkable nonadditivity in their effect on thermal stability. A simple model is proposed that relates this nonadditivity to the occurrence of independent partial unfolding processes that occur in parallel at elevated temperatures. To prove this model, a thermolysin-like protease was designed in which two mutations located ∼35 Å apart in the structure individually exert small stabilizing effects of 2.3 and 4.1 °C, respectively, but when combined stabilize the protease by 14.6 °C. This overadditivity, which follows directly from the model, confirms that unfolding of this engineered protease starts in parallel at two different regions of the protein.

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