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 similar to 35 Angstrom apart in the structure individually exert small stabilizing effects of 2.3 and 4.1 degrees C, respectively, but when combined stabilize the protease by 14.6 degrees 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.