PREDICTION AND ANALYSIS OF STRUCTURE, STABILITY AND UNFOLDING OF THERMOLYSIN-LIKE PROTEASES

Bacillus neutral proteases (NPs) form a group of well-characterized ho mologous enzymes, that exhibit large differences in thermostability. T he three-dimensional (3D) structures of several of these enzymes have been modelled on the basis of the crystal structures of the NPs of B. thermoproteolyticus (thermolysin) and B. cereus. Several new technique s have been developed to improve the model-building procedures. Also a 'model-building by mutagenesis' strategy was used, in which mutants w ere designed just to shed light on parts of the structures that were p articularly hard to model. The NP models have been used for the predic tion of site-directed mutations aimed at improving the thermostability of the enzymes. Predictions were made using several novel computation al techniques, such as position-specific rotamer searching, packing qu ality analysis and property-profile database searches. Many stabilizin g mutations were predicted and produced: improvement of hydrogen bondi ng, exclusion of buried water molecules, capping helices, improvement of hydrophobic interactions and entropic stabilization have been appli ed successfully. At elevated temperatures NPs are irreversibly inactiv ated as a result of autolysis. It has been shown that this denaturatio n process is independent of the protease activity and concentration an d that the inactivation follows first-order kinetics. From this it has been conjectured that local unfolding of (surface) loops, which rende rs the protein susceptible to autolysis, is the rate-limiting step. De spite the particular nature of the thermal denaturation process, norma l rules for protein stability can be applied to NPs. However, rather t han stabilizing the whole protein against global unfolding, only a sma ll region has to be protected against local unfolding. In contrast to proteins in general, mutational effects in proteases are not additive and their magnitude is strongly dependent on the location of the mutat ion. Mutations that alter the stability of the NP by a large amount ar e located in a relatively weak region (or more precisely, they affect a local unfolding pathway with a relatively low free energy of activat ion). One weak region, that is supposedly important in the early steps of NP unfolding, has been determined in the NP of B. stearothermophil us. After eliminating this weakest link a drastic increase in thermost ability was observed and the search for the second-weakest link, or th e second-lowest energy local unfolding pathway is now in progress. Hop efully, this approach can be used to unravel the entire early phase of unfolding.