Publication

THE EFFECT OF ENGINEERING SURFACE LOOPS ON THE THERMAL-STABILITY OF BACILLUS-SUBTILIS NEUTRAL PROTEASE

HARDY, F., VRIEND, G., VANDERVINNE, B., FRIGERIO, F., GRANDI, G., VENEMA, G. & EIJSINK, VGH., Mar-1994, In : Protein Engineering. 7, 3, p. 425-430 6 p.

Research output: Contribution to journalArticleAcademicpeer-review

  • F HARDY
  • G VRIEND
  • B VANDERVINNE
  • F FRIGERIO
  • G GRANDI
  • G VENEMA
  • VGH EIJSINK

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 degrees C, similar to 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.

Original languageEnglish
Pages (from-to)425-430
Number of pages6
JournalProtein Engineering
Volume7
Issue number3
Publication statusPublished - Mar-1994

    Keywords

  • BACILLUS, LOOP, NEUTRAL PROTEASE, SURFACE, THERMAL STABILITY, NUCLEOTIDE-SEQUENCE, LIMITED PROTEOLYSIS, BINDING LOOP, AMINO-ACID, THERMOLYSIN, GENE, STEAROTHERMOPHILUS, THERMOSTABILITY, RESOLUTION, EXPRESSION

ID: 6377402