INCREASING THE THERMOSTABILITY OF A NEUTRAL PROTEASE BY REPLACING POSITIVELY CHARGED AMINO-ACIDS IN THE N-TERMINAL TURN OF ALPHA-HELICES

Vincent G H Eijsink, Gerrit VRIEND, Bertus van den Burg, J Rob van der Zee, Gerard Venema

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Abstract

The 247-260 and 289-299 alpha-helices of Bacillus subtilis neutral protease have a lysine in their N-terminal turn. These lysines were replaced by Ser or Asp in order to improve electrostatic interactions with the alpha-helix dipole. After replacing Lys by Ser at positions 249 or 290, the thermostability of the enzyme was increased by 0.3 and 1.0-degrees-C, respectively. The Asp249 and Asp290 mutants exhibited a stabilization of 0.6 and 1.2-degrees-C, respectively. The results show the feasibility of stabilizing enzymes by introducing favourable residues at the end of alpha-helices.

Original languageEnglish
Pages (from-to)165-170
Number of pages6
JournalProtein Engineering
Volume5
Issue number2
DOIs
Publication statusPublished - Mar-1992

Keywords

  • ALPHA-HELIX
  • BACILLUS
  • HELIX DIPOLE
  • NEUTRAL PROTEASE
  • THERMOSTABILITY
  • BACILLUS-SUBTILIS
  • DIPOLE MODEL
  • PROTEINS
  • THERMOLYSIN
  • STABILITY
  • STABILIZATION
  • RESOLUTION
  • EXPRESSION
  • MUTATIONS
  • CLONING

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