title: The effect of engineering surface loops on the thermal stability of Bacillus subtilis neutral protease.
abstract: Using genetic techniques the contribution of surface loops to the thermal stability of Bacillus subtilis neutral protease (NP-sub) was studied. Mutations were designed to make the surface of NP-sub more similar to the surface of more thermostable neutral proteases such as thermolysin (TLN). The mutations included the replacement of an irregular loop by a shorter variant and the introduction of a ten-residue beta-hairpin. In general, these drastic mutations had little effect on the production and activity of NP-sub, indicating the feasibility of major structural rearrangements at the surface of proteins. In the most stable mutant, exhibiting an increase in thermal stability of 1.1 degree C, approximately 10% of the surface of NP-sub was modified. Several NP-sub variants carrying multiple mutations were constructed. Non-additive effects on thermal stability were observed, which were interpreted on the basis of a model for thermal inactivation, that emphasizes the importance of local unfolding processes for thermal stability.
authors.affiliation: Department of Genetics, University of Groningen, Haren, The Netherlands.
authors.forename: F
authors.initials: F
authors.lastname: Hardy
authors.forename: G
authors.initials: G
authors.lastname: Vriend
authors.forename: B
authors.initials: B
authors.lastname: van der Vinne
authors.forename: F
authors.initials: F
authors.lastname: Frigerio
authors.forename: G
authors.initials: G
authors.lastname: Grandi
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; D001426:Bacterial Proteins; D004795:Enzyme Stability; D006358:Hot Temperature; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013329:Structure-Activity Relationship; D013820:Thermolysin / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D008666:Metalloendopeptidases; D013820:Thermolysin; C071298:bacillolysin