The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease |
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Authors: | Eijsink, Vincent G.H. Dijkstra, Bauke W. Vriend, Gerrit van der Zee, J.Rob Vettman, Oene R. van der Vinne, B. van den Burg, B. Kempe, S. Venema, G. |
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Affiliation: | Department of Genetics, Centre for Biological Sciences, University of Groningen Kerklaan 30, 9751 NN Haren, The Netherlands 1EMBL, BIOcomputing program Meyerhofstrasse 1, 6900 Heidelberg, Germany 2BIOSON Research Institute, University of Groningen Nijenborgh 4, 9747 AG Groningen, The Netherlands |
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Abstract: | Cavities in the hydrophobic core of the neutral protease ofBacillus stearothermophilus were analyzed using a threedimensionalmodel that was inferred from the crystal structure of thermolysin,the highly homologous neutral protease of B.thermoproteolyticus(85% sequence identity). Sitedirected mutagenesis wasused to fill some of these cavities, thereby improving hydrophobicpacking in the protein interior. The mutations had small effectson the thermostability, even after drastic changes, such asLeu284Trp and Met168Trp. The effects on T50, the temperatureat which 50% of the enzyme is irreversibly inactivated in 30min, ranged from 0.0 to +0.4°C. These results can be explainedby assuming that the mutations have positive and negative structuraleffects of approximately the same magnitude. Alternatively,it could be envisaged that the local unfolding steps, whichrender the enzyme susceptible towards autolysis and which arerate limiting in the process of thermal inactivation, are onlyslightly affected by alterations in the hydrophobic core. |
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Keywords: | Bacillus/ cavity/ hydrophobic/ neutral protease/ thermostability |
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