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Protein stabilization by hydrophobic interactions at the surface

B Van den Burg¹, B W Dijkstra, G Vriend

¹Department of Genetics, University of Groningen, Haren, The Netherlands.

European Journal of Biochemistry

|March 15, 1994

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Site-directed mutagenesis of Bacillus stearothermophilus neutral protease revealed that specific amino acid substitutions at residue 63 significantly enhance thermal stability. Hydrophobic interactions at this surface-exposed site are key determinants of protein thermostability.

Area of Science:

Biochemistry
Protein Engineering
Enzyme Kinetics

Background:

Thermostable enzymes are crucial for industrial applications.
Understanding protein thermal stability is essential for protein engineering.
Bacillus stearothermophilus neutral protease is a model system for studying enzyme stability.

Purpose of the Study:

To investigate the role of solvent-exposed residue 63 in the thermal stability of Bacillus stearothermophilus neutral protease.
To identify amino acid substitutions that enhance the enzyme's thermostability.

Main Methods:

Site-directed mutagenesis was used to introduce twelve different amino acid substitutions at residue 63.
Thermal stability of the wild-type and mutant enzymes was assessed.
Analysis of the effects of mutations on enzyme stability.

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Main Results:

Introducing Arg, Lys, or bulky hydrophobic amino acids at residue 63 significantly increased thermal stability.
Hydrophobic contacts in the surface-located region were identified as major determinants of thermal stability.
Mutations demonstrated that surface residue contributions to stability can differ from general concepts.

Conclusions:

Residue 63 plays a critical role in the thermal stability of Bacillus stearothermophilus neutral protease.
Strategic amino acid substitutions, particularly those enhancing hydrophobic interactions, can be used to stabilize thermolysin-like proteases.
The findings offer new strategies for protein stabilization via site-directed mutagenesis.