Abstract
Many proteins lack the thermodynamic stability and/or solubility that is required for their use in a desired application. For this reason, it can be advantageous to improve these qualities through rational protein engineering. An effective means for achieving this goal is to use sequence alignment analysis to select amino acid substitutions that are likely to increase the thermodynamic stability or solubility of a protein. Advantages of using this approach are that generally only a small number of substitutions need to be tested, these substitutions are rarely debilitating to protein function, and knowledge of the three-dimensional structure of the protein of interest is not required. This chapter will describe approaches that have been used to exploit the information contained in sequence alignments for the engineering of improved protein properties.
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References
Eijsink, V. G., Bjork, A., Gaseidnes, S., Sirevag, R., Synstad, B., van den Burg, B., et al. (2004) Rational engineering of enzyme stability. J. Biotechnol. 113, 105–120.
Lehmann, M. and Wyss, M. (2001) Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution. Curr. Opin. Biotechnol. 12, 371–375.
van den Burg, B. and Eijsink, V. G. (2002) Selection of mutations for increased protein stability. Curr. Opin. Biotechnol. 13, 333–337.
Vieille, C. and Zeikus, G. J. (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol. Mol. Biol. Rev. 65, 1–43.
Arnold, F. H., Wintrode, P. L., Miyazaki, K., and Gershenson, A. (2001) How enzymes adapt: lessons from directed evolution. Trends Biochem. Sci. 26, 100–106.
Steipe, B., Schiller, B., Pluckthun, A., and Steinbacher, S. (1994) Sequence statis-tics reliably predict stabilizing mutations in a protein domain. J. Mol. Biol. 240, 188–192.
Wirtz, P. and Steipe, B. (1999) Intrabody construction and expression III: engi-neering hyperstable V(H) domains. Protein Sci. 8, 2245–2250.
Rath, A. and Davidson, A. R. (2000) The design of a hyperstable mutant of the Abp1p SH3 domain by sequence alignment analysis. Protein Sci. 9, 2457–2469.
Wang, Q., Buckle, A. M., Foster, N. W., Johnson, C. M., and Fersht, A. R. (1999) Design of highly stable functional GroEL minichaperones. Protein Sci. 8, 2186–2193.
Nikolova, P. V., Henckel, J., Lane, D. P., and Fersht, A. R. (1998) Semirational design of active tumor suppressor p53 DNA binding domain with enhanced stability. Proc. Natl. Acad. Sci. USA 95, 14675–14680.
Lehmann, M., Kostrewa, D., Wyss, M., Brugger, R., D’Arcy, A., Pasamontes, L., et al. (2000) From DNA sequence to improved functionality: using protein sequence comparisons to rapidly design a thermostable consensus phytase. Protein Eng. 13, 49–57.
Lehmann, M., Loch, C., Middendorf, A., Studer, D., Lassen, S. F., Pasamontes, L., et al. (2002) The consensus concept for thermostability engineering of pro-teins: further proof of concept. Protein Eng. 15, 403–411.
Jiang, X., Kowalski, J., and Kelly, J. W. (2001) Increasing protein stability using a rational approach combining sequence homology and structural alignment: sta-bilizing the WW domain. Protein Sci. 10, 1454–1465.
Mosavi, L. K., Minor, D. L., Jr., and Peng, Z. Y. (2002) Consensus-derived structural determinants of the ankyrin repeat motif. Proc. Natl. Acad. Sci. USA 99, 16029–16034.
Ito, T. and Wagner, G. (2004) Using codon optimization, chaperone co-expression, and rational mutagenesis for production and NMR assignments of human eIF2 alpha. J. Biomol. NMR 28, 357–367.
Malissard, M. and Berger, E. G. (2001) Improving solubility of catalytic domain of human beta-1,4-galactosyltransferase 1 through rationally designed amino acid replacements. Eur. J. Biochem. 268, 4352–4358.
Sun, Z. Y., Dotsch, V., Kim, M., Li, J., Reinherz, E. L., and Wagner, G. (1999) Functional glycan-free adhesion domain of human cell surface receptor CD58: design, production and NMR studies. EMBO J. 18, 2941–2949.
Irving, J. A., Askew, D. J., and Whisstock, J. C. (2004) Computational analysis of evolution and conservation in a protein superfamily. Methods 32, 73–92.
Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673–4680.
Clamp, M., Cuff, J., Searle, S. M., and Barton, G. J. (2004) The Jalview Java alignment editor. Bioinformatics 20, 426–427.
Bateman, A., Birney, E., Durbin, R., Eddy, S. R., Howe, K. L., and Sonnhammer, E. L. (2000) The Pfam protein families database. Nucleic Acids Res. 28, 263–266.
Schultz, J., Milpetz, F., Bork, P., and Ponting, C. P. (1998) SMART, a simple modular architecture research tool: identification of signaling domains. Proc. Natl. Acad. Sci. USA 95, 5857–5864.
Sander, C. and Schneider, R. (1991) Database of homology-derived protein structures and the structural meaning of sequence alignment. Proteins 9, 56–68.
Henikoff, S. and Henikoff, J. G. (1994) Position-based sequence weights. J. Mol. Biol. 243, 574–578.
Lewis, H. A., Zhao, X., Wang, C., Sauder, J. M., Rooney, I., Noland, B. W., et al. (2005) Impact of the deltaF508 mutation in first nucleotide-binding domain of human cystic fibrosis transmembrane conductance regulator on domain folding and structure. J. Biol. Chem. 280, 1346–1353.
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Davidson, A.R. (2006). Multiple Sequence Alignment as a Guideline for Protein Engineering Strategies. In: Guerois, R., de la Paz, M.L. (eds) Protein Design. Methods in Molecular Biology, vol 340. Humana Press. https://doi.org/10.1385/1-59745-116-9:171
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DOI: https://doi.org/10.1385/1-59745-116-9:171
Publisher Name: Humana Press
Print ISBN: 978-1-58829-585-9
Online ISBN: 978-1-59745-116-1
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