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Computational design of the Fyn SH3 domain with increased stability through optimization of surface charge–charge interactions
Author(s) -
Schweiker Katrina L.,
ZarrineAfsar Arash,
Davidson Alan R.,
Makhatadze George I.
Publication year - 2007
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.073091607
Subject(s) - thermostability , fyn , circular dichroism , protein design , sh3 domain , surface charge , charge density , protein stability , chemistry , crystallography , differential scanning calorimetry , protein secondary structure , protein structure , chemical physics , materials science , physics , thermodynamics , proto oncogene tyrosine protein kinase src , biochemistry , enzyme , quantum mechanics , kinase
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 β‐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, Δ T m = 12.3 ± 0.2°C, and stability, ΔΔ 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.