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Protein Science (2007), 16:2694-2702. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Computational design of the Fyn SH3 domain with increased stability through optimization of surface charge–charge interactions

Katrina L. Schweiker1,2, Arash Zarrine-Afsar3, Alan R. Davidson3,4, and George I. Makhatadze2

1 Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, USA
2 Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180–3590, USA
3 Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
4 Department of Biochemistry, University of Toronto, Toronto, Ontario M5S 1A8, Canada

(RECEIVED June 24, 2007; FINAL REVISION August 29, 2007; ACCEPTED August 29, 2007)

Computational design of surface charge–charge interactions has been demonstrated to be an effective way to increase both the thermostability and the stability of proteins. To test the robustness of this approach for proteins with predominantly beta-sheet secondary structure, the chicken isoform of the Fyn SH3 domain was used as a model system. Computational analysis of the optimal distribution of surface charges showed that the increase in favorable energy per substitution begins to level off at five substitutions; hence, the designed Fyn sequence contained four charge reversals at existing charged positions and one introduction of a new charge. Three additional variants were also constructed to explore stepwise contributions of these substitutions to Fyn stability. The thermodynamic stabilities of the variants were experimentally characterized using differential scanning calorimetry and far-UV circular dichroism spectroscopy and are in very good agreement with theoretical predictions from the model. The designed sequence was found to have increased the melting temperature, {Delta}T m = 12.3 ± 0.2°C, and stability, {Delta}{Delta}G(25°C) = 7.1 ± 2.2 kJ/mol, relative to the wild-type protein. The experimental data suggest that a significant increase in stability can be achieved through a very small number of amino acid substitutions. Consistent with a number of recent studies, the presented results clearly argue for a seminal role of surface charge–charge interactions in determining protein stability and suggest that the optimization of surface interactions can be an attractive strategy to complement algorithms optimizing interactions in the protein core to further enhance protein stability.

Keywords: rational design; protein stability; charge–charge interactions; protein surface


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