MUTATIONAL ANALYSIS OF A SURFACE-AREA THAT IS CRITICAL FOR THE THERMAL-STABILITY OF THERMOLYSIN-LIKE PROTEASES

Site-directed mutagenesis was used to assess the contribution of indiv idual residues and a bound calcium in the 55-69 region of the thermoly sin-like protease of Bacillus stearothermophilus (TLP-ste) to thermal stability. The importance of the 55-69 region was reflected by finding that almost all mutations had drastic effects on stability. These eff ects (both stabilizing and destabilizing) were obtained by mutations a ffecting main chain flexibility, as well as by mutations affecting the interaction between the 55-69 region and the rest of the protease mol ecule, The calcium-dependency of stability could be largely abolished by mutating one of its ligands (Asp57 or Asp59). In the case of the As p57-->Ser mutation the accompanying loss in stability was modest compa red with the effects of other destabilizing mutations or the effects o f (combinations of) stabilizing mutations, The detailed knowledge of t he stability-determining region of TLP-ste permits effective rational design of stabilizing mutations, which, presumably, are also useful fo r related TLP such as thermolysin, This is demonstrated by the success ful design of a stabilizing salt bridge involving residues 65 and 11.