title: Structural features of neutral protease from Bacillus subtilis deduced from model-building and limited proteolysis experiments.
abstract: The overall folding of neutral protease from Bacillus subtilis has been predicted by computer-aided modelling, taking as a basis the known three-dimensional structure of thermolysin. As expected from the 50% similarity of sequence between the two proteins, the structure of B. subtilis protease is similar to that of thermolysin, including the two-domain topology and location of elements of regular secondary structure (helices and strands), whereas specific differences were predicted in loop regions. A protruding and loose loop predicted in B. subtilis has been detected also experimentally by a limited proteolysis approach. Incubation of B. subtilis protease at pH 9.0 for 24 h at room temperature with trypsin at 20:1 ratio (by mass) leads to a specific and almost quantitative fission of the Arg214-Asn215 peptide bond located in a highly exposed, and thus probably flexible, loop of the protease. On the other hand, thermolysin was completely resistant to tryptic hydrolysis when reacted under identical conditions. The 'nicked' B. subtilis protease can be isolated by gel filtration chromatography at neutral pH, whereas the two constituting fragments 1-214 and 215-300 are separated under protein-denaturing conditions. Overall, these results indicate that the limited proteolysis approach can pinpoint a peculiar difference in surface structure between the two similar protein molecules of B. subtilis neutral protease and thermolysin and emphasize the potential use of proteolytic enzymes as structural probes of globular proteins.
authors.affiliation: Department of Organic Chemistry, University of Padua, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Signor
authors.forename: C
authors.initials: C
authors.lastname: Vita
authors.forename: A
authors.initials: A
authors.lastname: Fontana
authors.forename: F
authors.initials: F
authors.lastname: Frigerio
authors.forename: M
authors.initials: M
authors.lastname: Bolognesi
authors.forename: S
authors.initials: S
authors.lastname: Toma
authors.forename: R
authors.initials: R
authors.lastname: Gianna
authors.forename: E
authors.initials: E
authors.lastname: De Gregoriis
authors.forename: G
authors.initials: G
authors.lastname: Grandi
mesh_terms: D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D002850:Chromatography, Gel; D003198:Computer Simulation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000235:genetics*; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D013820:Thermolysin / Q000235:genetics; D014357:Trypsin
chemical_list: D010446:Peptide Fragments; D014357:Trypsin; D015260:Neprilysin; D013820:Thermolysin