Structural features of neutral protease from Bacillus subtilis deduced from model-building and limited proteolysis experiments

G. Signor, C. Vita, A. Fontana, F. Frigerio, M. Bolognesi, S. Toma, R. Gianna, E. De Gregoriis, G. Grandi

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Abstract

The overall folding of neutral protease from Bacillus subtilis has been prediced 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 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 be pinpoint a peculiar difference in surface structure between the two similar protien molecules of B. subtilis neutral protease and thermolysin and emphasize the potential use of proteolytic enzymes as structural probes of globular proteins.

Original languageEnglish
Pages (from-to)221-227
Number of pages7
JournalEuropean Journal of Biochemistry
Volume189
Issue number2
Publication statusPublished - 1990

ASJC Scopus subject areas

  • Biochemistry

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    Signor, G., Vita, C., Fontana, A., Frigerio, F., Bolognesi, M., Toma, S., Gianna, R., De Gregoriis, E., & Grandi, G. (1990). Structural features of neutral protease from Bacillus subtilis deduced from model-building and limited proteolysis experiments. European Journal of Biochemistry, 189(2), 221-227.