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Protein stabilization by hydrophobic interactions at the

By Vincent G. H. Eijsink

Abstract

The contribution of the solvent-exposed residue 63 to thermal stability of the thermolysin-like neutral protease of Bacillus stearothermophilus was studied by analyzing the effect of twelve different amino acid substitutions at this position. The thermal stability of the enzyme was increased considerably by introducing Arg, Lys or bulky hydrophobic amino acids. In general, the effects of the mutations showed that hydrophobic contacts in this surface-located region of the protein are a major determinant of thermal stability. This observation contrasts with general concepts concerning the contribution of surface-located residues and surface hydrophobicity to protein stability and indicates new ways for protein stabilization by site-directed mutagenesis. Bacillus neutral proteases (NP) are metalloendopeptidases that resemble thermolysin, the well-characterized extremely thermostable NP of Bacillus stearothermophilus. The crystal structures of thermolysin (Matthews et al., 1972; Holmes and Matthews, 1982) and of the NP from Bacillus cereus (Pauptit et al., 1988; Stark et al., 1992) have been elucidated, and on the basis of these structures three-dimensional models of other NP have been built (Signor et al., 1990; Vriend and Eijsink, 1993). Within the family of NP large differences in thermal stability occur and therefore these enzymes form an interesting system to study structurestability relationships. Recent protein-engineering studies have revealed some of the structural factors that determine the thermal stability of NP (Imanaka et al., 1986; Toma e

Year: 1993
OAI identifier: oai:CiteSeerX.psu:10.1.1.318.4084
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