title: Model building of a thermolysin-like protease by mutagenesis.
abstract: The present study concerns the use of site-directed mutagenesis experiments to optimize a three-dimensional model of the neutral protease of Bacillus subtilis (NP-sub). An initial model of NP-sub was constructed using the crystal structures of the homologous neutral proteases of Bacillus thermoproteolyticus (thermolysin) and Bacillus cereus as templates. The largest portion of NP-sub could be modelled satisfactorily, using standard techniques, but several surface-located regions could only be modelled with a high degree of uncertainty. In order to make the model more reliable in these regions a 'model building by mutagenesis' approach was adopted. Mutations were designed such that their effect on thermal stability could indicate how their local environment should be modelled. This approach provided insight in the local structure of several regions in NP-sub that were hard to model on the basis of homology with the two known structures alone.
authors.affiliation: Eniricerche, San Donato Milanese, MI, Italy.
authors.forename: F
authors.initials: F
authors.lastname: Frigerio
authors.forename: I
authors.initials: I
authors.lastname: Margarit
authors.forename: R
authors.initials: R
authors.lastname: Nogarotto
authors.forename: G
authors.initials: G
authors.lastname: Grandi
authors.forename: G
authors.initials: G
authors.lastname: Vriend
authors.forename: F
authors.initials: F
authors.lastname: Hardy
authors.forename: O R
authors.initials: OR
authors.lastname: Veltman
authors.forename: G
authors.initials: G
authors.lastname: Venema
authors.forename: V G
authors.initials: VG
authors.lastname: Eijsink
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis; D001426:Bacterial Proteins; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D016415:Sequence Alignment
chemical_list: D001426:Bacterial Proteins; D008666:Metalloendopeptidases; C071298:bacillolysin