The effect of engineering surface loops on the thermal stability of Bacillus subtilis neutral protease.
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.
Hardy
F
F
Department of Genetics, University of Groningen, Haren, The Netherlands.
Vriend
G
G
van der Vinne
B
B
Frigerio
F
F
Grandi
G
G
Venema
G
G
Eijsink
V G
VG
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
D001426:Bacterial Proteins; D010446:Peptide Fragments; D008666:Metalloendopeptidases; D013820:Thermolysin; C071298:bacillolysin