Enhancement of protein stability by the combination of point mutations in T4 lysozyme is additive |
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Authors: | Zhang, Xue-jun Baase, Walter A. Shoichet, Brian K. Wilson, Keith P. Matthews, Brian W. |
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Affiliation: | Institute of Molecular Biology, Howard Hughes Medical Institute and Department of Physics, University of Oregon Eugene, OR 97403, USA 1Protein Studies Program, Oklahoma Medical Research Foundation 825 NE 13th Street, Oklahoma City, OK 73104, USA 2Vertex Pharmaceuticals Inc. 40 Allston Street, Cambridge, MA 02139, USA |
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Abstract: | A number of mutations have been shown previously to stabilizeT4 lysozyme. By combining up to seven such mutations in thesame protein, the melting temperature was incrementally increasedby up to 83°C at pH 5.4 (![{Delta}](http://peds.oxfordjournals.org/math/Delta.gif) G = 3.6 kcal/mol). This shows thatit is possible to engineer a protein of enhanced thermostabilityby combining a series of rationally designed point mutations.It is also shown that this stabilization is achieved with onlyminor, localized changes in the structure of the protein. Thisis consistent with the observation that the change in stabilityof each of the multiple mutants is, in each case, additive,i.e. equal to the sum of the stability changes associated withthe constituent single mutants. One of the seven substitutions,Asn116 Asp, changes a residue that participates in substratebinding; not surprisingly, it causes a significant loss in activity.Ignoring this mutation, there is a gradual reduction in activityas successively more mutations are combined. |
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Keywords: | additivity/ genetic engineering/ lysozyme/ protein stabilization/ thermostability |
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