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Crystal structures of thermally stable adenylate kinase mutants designed by local structural entropy optimization and structure-guided mutagenesis

Journal of the Korean Society for Applied Biological Chemistry volume 57, pages 661–665 (2014)Cite this article

Abstract

Thermally stable proteins are desirable in many industrial and laboratory settings, and numerous approaches have been developed to redesign proteins for higher thermal stability. Here, we report the crystal structures of two thermally stable adenylate kinase (AK) mutants that were designed by applying a combination of local structural entropy (LSE) optimization and structure-guided mutagenesis. Structure-guided mutagenesis resulted in stabilizing interactions connecting distant regions of the LSEoptimized AK sequence. This demonstrates the feasibility and importance of simultaneous optimization of local and global features in protein thermal stabilization. An additional AK mutant showed that small changes in side-chain configuration can greatly impact thermal stability.

References

  • Bae E and Phillips GN, Jr.. (2004) Structures and analysis of highly homologous psychrophilic, mesophilic, and thermophilic adenylate kinases. J Biol Chem 279, 28202–8.

    Article  CAS  Google Scholar 

  • Bae E and Phillips GN, Jr.. (2005) Identifying and engineering ion pairs in adenylate kinases. Insights from molecular dynamics simulations of thermophilic and mesophilic homologues. J Biol Chem 280, 30943–8.

    Article  CAS  Google Scholar 

  • Bae E and Phillips GN, Jr.. (2006) Roles of static and dynamic domains in stability and catalysis of adenylate kinase. Proc Natl Acad Sci U S A 103, 2132–7.

    Article  CAS  Google Scholar 

  • Bae E, Bannen RM, and Phillips GN, Jr.. (2008) Bioinformatic method for protein thermal stabilization by structural entropy optimization. Proc Natl Acad Sci U S A 105, 9594–7.

    Article  CAS  Google Scholar 

  • Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, and Bourne PE. (2000) The Protein Data Bank. Nucleic Acids Res 28, 235–42.

    Article  CAS  Google Scholar 

  • Chan CH, Liang HK, Hsiao NW, Ko MT, Lyu PC, and Hwang JK. (2004) Relationship between local structural entropy and protein thermostability. Proteins 57, 684–91.

    Article  CAS  Google Scholar 

  • Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, and Richardson DC. (2010) MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr 66, 12–21.

    Article  CAS  Google Scholar 

  • Daniel RM and Danson MJ. (2010) A new understanding of how temperature affects the catalytic activity of enzymes. Trends Biochem Sci 35, 584–91.

    Article  CAS  Google Scholar 

  • Eijsink VG, Bjork A, Gaseidnes S, Sirevag R, Synstad B, van den Burg B, and Vriend G. (2004) Rational engineering of enzyme stability. J Biotechnol 113, 105–20.

    Article  CAS  Google Scholar 

  • Emsley P and Cowtan K. (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126–32.

    Article  Google Scholar 

  • Liszka MJ, Clark ME, Schneider E, and Clark DS. (2012) Nature versus nurture: developing enzymes that function under extreme conditions. Annu Rev Chem Biomol Eng 3, 77–102.

    Article  CAS  Google Scholar 

  • McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, and Read RJ. (2007) Phaser crystallographic software. J Appl Crystallogr 40, 658–74.

    Article  CAS  Google Scholar 

  • Moon S, Bannen RM, Rutkoski TJ, Phillips GN Jr., and Bae E (2014a) Effectiveness and limitations of local structural entropy optimization in the thermal stabilization of mesophilic and thermophilic adenylate kinases. Proteins DOI: 10.1002/prot.24627.

    Google Scholar 

  • Moon S, Jung DK, Phillips GN Jr., and Bae E (2014b) An integrated approach for thermal stabilization of a mesophilic adenylate kinase. Proteins 82, 1947–59.

    Article  CAS  Google Scholar 

  • Murshudov GN, Vagin AA, and Dodson EJ. (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53, 240–55.

    Article  CAS  Google Scholar 

  • Otwinowski Z and Minor W. (1997) Processing of X-ray diffraction data collected in oscillation mode. Method Enzymol 276, 307–26.

    Article  CAS  Google Scholar 

  • Vagin A and Teplyakov A. (2010) Molecular replacement with MOLREP. Acta Crystallogr D Biol Crystallogr 66, 22–5.

    Article  CAS  Google Scholar 

Download references

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Authors and Affiliations

  1. Department of Agricultural Biotechnology, Seoul National University, Seoul, 151-921, Republic of Korea

    Sojin Moon

  2. Department of Agricultural Biotechnology, Center for Food and Bioconvergence, and Research Institute of Agriculture and Life Sciences, Seoul National University, Seoul, 151-921, Republic of Korea

    Euiyoung Bae

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  1. Sojin Moon
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  2. Euiyoung Bae
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Correspondence to Euiyoung Bae.

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Moon, S., Bae, E. Crystal structures of thermally stable adenylate kinase mutants designed by local structural entropy optimization and structure-guided mutagenesis. J Korean Soc Appl Biol Chem 57, 661–665 (2014). https://doi.org/10.1007/s13765-014-4228-4

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  • DOI: https://doi.org/10.1007/s13765-014-4228-4

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