References

The following references illustrate some of the many examples in which computational protein design (CPD) has been used to engineer proteins with new or improved properties. Also listed are studies in which computational tools have served to complement or enhance evolutionary engineering strategies. Recent reviews are also provided that summarize advances in CPD and describe applications in biocatalysis and therapeutics.



Reviews

Chen, T.S. & Keating, A.E. (2012)
Designing specific protein-protein interactions using computation, experimental library screening, or integrated methods. Protein Sci 21, 949–963

Pantazes, R.J., Grisewood, M.J. & Maranas, C.D. (2011)
Recent advances in computational protein design. Curr Opin Struc Biol 21, 467–472

Saven, J.G. (2011)
Computational protein design: engineering molecular diversity, nonnatural enzymes, nonbiological cofactor complexes, and membrane proteins. Curr Opin Chem Biol 15, 452–457

Morin, A., Meiler, J. & Mizoue, L.S. (2011)
Computational design of protein-ligand interfaces: potential in therapeutic development. Trends Biotechnol 29, 159–166

Karanicolas, J. & Kuhlman, B. (2009)
Computational design of affinity and specificity at protein-protein interfaces. Curr Opin Struc Biol 19, 458–463

Damborsky, J. & Brezovsky, J. (2009)
Computational tools for designing and engineering biocatalysts. Curr Opin Chem Biol 13, 26–34

Alvizo, O., Allen, B.D. & Mayo, S.L. (2007)
Computational protein design promises to revolutionize protein engineering. BioTechniques 42, 31–33– 35

Lazar, G.A., Marshall, S.A., Plecs, J.J., Mayo, S.L. & Desjarlais, J.R. (2003)
Designing proteins for therapeutic applications. Curr Opin Struc Biol 13, 513–518



Engineering New or Improved Properties

Enhancing stability

Le, Q.A.T., Joo, J.C., Yoo, Y.J. & Kim, Y.H. (2012)
Development of thermostable Candida antarctica lipase B through novel in silico design of disulfide bridge. Biotechnol Bioeng 109, 867–876

Miklos, A.E., Kluwe, C., Der, B.S., Pai, S., Sircar, A., Hughes, R.A., Berrondo, M., Xu, J., Codrea, V., Buckley, P.E., Calm, A.M., Welsh, H.S., Warner, C.R., Zacharko, M.A., Carney, J.P., Gray, J.J., Georgiou, G., Kuhlman, B. & Ellington, A.D. (2012)
Structure-based design of supercharged, highly thermoresistant antibodies. Chem Biol 19, 449–455

Diaz, J.E., Lin, C.-S., Kunishiro, K., Feld, B.K., Avrantinis, S.K., Bronson, J., Greaves, J., Saven, J.G. & Weiss, G.A. (2011)
Computational design and selections for an engineered, thermostable terpene synthase. Protein Sci 20, 1597–1606

Joo, J.C., Pack, S.P., Kim, Y.H. & Yoo, Y.J. (2011)
Thermostabilization of Bacillus circulans xylanase: computational optimization of unstable residues based on thermal fluctuation analysis. J Biotechnol 151, 56–65

Allen, B.D., Nisthal, A. & Mayo, S.L. (2010)
Experimental library screening demonstrates the successful application of computational protein design to large structural ensembles. Proc Natl Acad Sci USA 107, 19838–19843

Joo, J.C., Pohkrel, S., Pack, S.P. & Yoo, Y.J. (2010)
Thermostabilization of Bacillus circulans xylanase via computational design of a flexible surface cavity. J Biotechnol 146, 31–39

Kim, H.S., Le, Q.A.T. & Kim, Y.H. (2010)
Development of thermostable lipase B from Candida antarctica (CalB) through in silico design employing B-factor and RosettaDesign. Enzyme Microb Technol 47, 1–5

Kim, S.J., Lee, J.A., Joo, J.C., Yoo, Y.J., Kim, Y.H. & Song, B.K. (2010)
The development of a thermostable CiP (Coprinus cinereus peroxidase) through in silico design. Biotechnol Progress 26, 1038–1046

Gao, D., Narasimhan, D.L., Macdonald, J., Brim, R., Ko, M.-C., Landry, D.W., Woods, J.H., Sunahara, R.K. & Zhan, C.-G. (2009)
Thermostable variants of cocaine esterase for long-time protection against cocaine toxicity. Mol. Pharmacol. 75, 318–323

Gribenko, A.V., Patel, M.M., Liu, J., McCallum, S.A., Wang, C. & Makhatadze, G.I. (2009)
Rational stabilization of enzymes by computational redesign of surface charge-charge interactions. Proc Natl Acad Sci USA 106, 2601–2606

Dantas, G., Corrent, C., Reichow, S.L., Havranek, J.J., Eletr, Z.M., Isern, N.G., Kuhlman, B., Varani, G., Merritt, E.A. & Baker, D. (2007)
High-resolution structural and thermodynamic analysis of extreme stabilization of human procarboxypeptidase by computational protein design. J Mol Biol 366, 1209–1221

Schweiker, K.L., Zarrine-Afsar, A., Davidson, A.R. & Makhatadze, G.I. (2007)
Computational design of the Fyn SH3 domain with increased stability through optimization of surface charge charge interactions. Protein Sci 16, 2694–2702

Shah, P.S., Hom, G.K., Ross, S.A., Lassila, J.K., Crowhurst, K.A. & Mayo, S.L. (2007)
Full-sequence computational design and solution structure of a thermostable protein variant. J Mol Biol 372, 1–6

Strickler, S.S., Gribenko, A.V., Gribenko, A.V., Keiffer, T.R., Tomlinson, J., Reihle, T., Loladze, V.V. & Makhatadze, G.I. (2006)
Protein stability and surface electrostatics: a charged relationship. Biochemistry 45, 2761–2766

Zollars, E.S., Marshall, S.A. & Mayo, S.L. (2006)
Simple electrostatic model improves designed protein sequences. Protein Sci 15, 2014–2018

Korkegian, A., Black, M.E., Baker, D. & Stoddard, B.L. (2005)
Computational thermostabilization of an enzyme. Science 308, 857–860

Bolon, D.N., Marcus, J.S., Ross, S.A. & Mayo, S.L. (2003)
Prudent modeling of core polar residues in computational protein design. J Mol Biol 329, 611–622

Dantas, G., Kuhlman, B., Callender, D., Wong, M. & Baker, D. (2003)
A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins. J Mol Biol 332, 449–460

Filikov, A.V., Hayes, R.J., Luo, P., Stark, D.M., Chan, C., Kundu, A. & Dahiyat, B.I. (2002)
Computational stabilization of human growth hormone. Protein Sci 11, 1452–1461

Luo, P., Hayes, R.J., Chan, C., Stark, D.M., Hwang, M.Y., Jacinto, J.M., Juvvadi, P., Chung, H.S., Kundu, A., Ary, M.L. & Dahiyat, B.I. (2002)
Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screening. Protein Sci 11, 1218–1226

Spector, S., Wang, M., Carp, S.A., Robblee, J., Hendsch, Z.S., Fairman, R., Tidor, B. & Raleigh, D.P. (2000)
Rational modification of protein stability by the mutation of charged surface residues. Biochemistry 39, 872–879

Malakauskas, S.M. & Mayo, S.L. (1998)
Design, structure and stability of a hyperthermophilic protein variant. Nat Struct Biol 5, 470–475

Enhancing solubility

Slovic, A.M., Kono, H., Lear, J.D., Saven, J.G. & DeGrado, W.F. (2004)
Computational design of water-soluble analogues of the potassium channel KcsA. Proc Natl Acad Sci USA 101, 1828–1833

Slovic, A.M., Summa, C.M., Lear, J.D. & DeGrado, W.F. (2003)
Computational design of a water-soluble analog of phospholamban. Protein Sci 12, 337–348

Altering pH profile

Beliën, T., Joye, I.J., Delcour, J.A. & Courtin, C.M. (2009)
Computational design-based molecular engineering of the glycosyl hydrolase family 11 B. subtilis XynA endoxylanase improves its acid stability. Protein Eng Des Sel 22, 587–596

Improving enzymatic activity

Khersonsky, O., Röthlisberger, D., Wollacott, A.M., Murphy, P., Dym, O., Albeck, S., Kiss, G., Houk, K.N., Baker, D. & Tawfik, D.S. (2011)
Optimization of the in-silico-designed kemp eliminase KE70 by computational design and directed evolution. J Mol Biol 407, 391–412

Lassila, J.K., Keeffe, J.R., Oelschlaeger, P. & Mayo, S.L. (2005)
Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity. Protein Eng Des Sel 18, 161–163

Oelschlaeger, P. & Mayo, S.L. (2005)
Hydroxyl groups in the ββ-sandwich of metallo-β-lactamases favor enzyme activity: a computational protein design study. J Mol Biol 350, 395–401

Designing enzymes de novo

Khare, S.D., Kipnis, Y., Greisen, P., Takeuchi, R., Ashani, Y., Goldsmith, M., Song, Y., Gallaher, J.L., Silman, I., Leader, H., Sussman, J.L., Stoddard, B.L., Tawfik, D.S. & Baker, D. (2012)
Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis. Nat Chem Biol 8, 294–300

Privett, H.K., Kiss, G., Lee, T.M., Blomberg, R., Chica, R.A., Thomas, L.M., Hilvert, D., Houk, K.N. & Mayo, S.L. (2012)
Iterative approach to computational enzyme design. Proc Natl Acad Sci USA 109, 3790–3795

Siegel, J.B., Zanghellini, A., Lovick, H.M., Kiss, G., Lambert, A.R., St Clair, J.L., Gallaher, J.L., Hilvert, D., Gelb, M.H., Stoddard, B.L., Houk, K.N., Michael, F.E. & Baker, D. (2010)
Computational design of an enzyme catalyst for a stereoselective bimolecular Diels-Alder reaction. Science 329, 309–313

Jiang, L., Althoff, E.A., Clemente, F.R., Doyle, L., Röthlisberger, D., Zanghellini, A., Gallaher, J.L., Betker, J.L., Tanaka, F., Barbas, C.F., Hilvert, D., Houk, K.N., Stoddard, B.L. & Baker, D. (2008)
De novo computational design of retro-aldol enzymes. Science 319, 1387–1391

Röthlisberger, D., Khersonsky, O., Wollacott, A.M., Jiang, L., DeChancie, J., Betker, J., Gallaher, J.L., Althoff, E.A., Zanghellini, A., Dym, O., Albeck, S., Houk, K.N., Tawfik, D.S. & Baker, D. (2008)
Kemp elimination catalysts by computational enzyme design. Nature 453, 190–195

Altering binding specificity/affinity 

Alvizo, O., Mittal, S., Mayo, S.L. & Schiffer, C.A. (2012)
Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease. Protein Sci 21, 1029–1041

Allen, B.D. & Mayo, S.L. (2010)
An efficient algorithm for multistate protein design based on FASTER. J Comput Chem 31, 904–916

Frey, K.M., Georgiev, I., Donald, B.R. & Anderson, A.C. (2010)
Predicting resistance mutations using protein design algorithms. Proc Natl Acad Sci USA 107, 13707–13712

Lippow, S.M., Moon, T.S., Basu, S., Yoon, S.-H., Li, X., Chapman, B.A., Robison, K., Lipovšek, D. & Prather, K.L.J. (2010)
Engineering enzyme specificity using computational design of a defined-sequence library. Chem Biol 17, 1306–1315

Murphy, P.M., Bolduc, J.M., Gallaher, J.L., Stoddard, B.L. & Baker, D. (2009)
Alteration of enzyme specificity by computational loop remodeling and design. PNAS 106, 9215–9220

Yosef, E., Politi, R., Choi, M.H. & Shifman, J.M. (2009)
Computational design of calmodulin mutants with up to 900-fold increase in binding specificity. J Mol Biol 385, 1470–1480

Ashworth, J., Havranek, J.J., Duarte, C.M., Sussman, D., Monnat, R.J., Stoddard, B.L. & Baker, D. (2006)
Computational redesign of endonuclease DNA binding and cleavage specificity. Nature 441, 656–659

Shifman, J.M. & Mayo, S.L. (2003)
Exploring the origins of binding specificity through the computational redesign of calmodulin. Proc Natl Acad Sci USA 100, 13274–13279

Shifman, J.M. & Mayo, S.L. (2002)
Modulating calmodulin binding specificity through computational protein design. J Mol Biol 323, 417–423

Designing new or specific protein-protein interfaces

Chen, T.S. & Keating, A.E. (2012)
Designing specific protein-protein interactions using computation, experimental library screening, or integrated methods. Protein Sci 21, 949–963

Jha, R.K., Leaver-Fay, A., Yin, S., Wu, Y., Butterfoss, G.L., Szyperski, T., Dokholyan, N.V. & Kuhlman, B. (2010)
Computational design of a PAK1 binding protein. J Mol Biol 400, 257–270

Grigoryan, G., Reinke, A.W. & Keating, A.E. (2009)
Design of protein-interaction specificity gives selective bZIP-binding peptides. Nature 458, 859–864

Huang, P.-S., Love, J.J. & Mayo, S.L. (2007)
A de novo designed protein protein interface. Protein Sci 16, 2770–2774

Improving spectral properties

Chica, R.A., Moore, M.M., Allen, B.D. & Mayo, S.L. (2010)
Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries. Proc Natl Acad Sci USA 107, 20257–20262

Mena, M.A., Treynor, T.P., Mayo, S.L. & Daugherty, P.S. (2006)
Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library. Nat Biotechnol 24, 1569–1571

Stabilizing target conformational states

Luo, B.-H., Karanicolas, J., Harmacek, L.D., Baker, D. & Springer, T.A. (2009)
Rationally designed integrin β3 mutants stabilized in the high affinity conformation. J Biol Chem 284, 3917–3924

Shimaoka, M., Shifman, J.M., Jing, H., Takagi, J., Mayo, S.L. & Springer, T.A. (2000)
Computational design of an integrin I domain stabilized in the open high affinity conformation. Nat Struct Biol 7, 674–678

Creating sequences that can switch between conformational states

Ambroggio, X.I. & Kuhlman, B. (2006)
Computational design of a single amino acid sequence that can switch between two distinct protein folds. J Am Chem Soc 128, 1154–1161



Enhancing Protein Engineering

Designing degenerate codon libraries

Allen, B.D., Nisthal, A. & Mayo, S.L. (2010)
Experimental library screening demonstrates the successful application of computational protein design to large structural ensembles. Proc Natl Acad Sci USA 107, 19838–19843

Designing focused combinatorial libraries

Allen, B.D., Nisthal, A. & Mayo, S.L. (2010)
Experimental library screening demonstrates the successful application of computational protein design to large structural ensembles. Proc Natl Acad Sci USA 107, 19838–19843

Guntas, G., Purbeck, C. & Kuhlman, B. (2010)
Engineering a protein-protein interface using a computationally designed library. PNAS 107, 19296–19301

Optimizing directed evolution

Voigt, C.A., Mayo, S.L., Arnold, F.H. & Wang, Z.G. (2001)
Computational method to reduce the search space for directed protein evolution. Proc Natl Acad Sci USA 98, 3778–3783

Improving compatibility and diversity of recombination experiments

Heinzelman, P., Snow, C.D., Wu, I., Nguyen, C., Villalobos, A., Govindarajan, S., Minshull, J. & Arnold, F.H. (2009)
A family of thermostable fungal cellulases created by structure-guided recombination. Proc Natl Acad Sci USA 106, 5610–5615

Meyer, M.M., Hochrein, L. & Arnold, F.H. (2006)
Structure-guided SCHEMA recombination of distantly related β-lactamases. Protein Eng Des Sel 19, 563–570

Otey, C.R., Landwehr, M., Endelman, J.B., Hiraga, K., Bloom, J.D. & Arnold, F.H. (2006)
Structure-guided recombination creates an artificial family of cytochromes P450. PLoS Biol 4, e112

Voigt, C.A., Martinez, C., Wang, Z.-G., Mayo, S.L. & Arnold, F.H. (2002)
Protein building blocks preserved by recombination. Nat Struct Biol 9, 553–558

Creating new activity that can be improved with directed evolution

Privett, H.K., Kiss, G., Lee, T.M., Blomberg, R., Chica, R.A., Thomas, L.M., Hilvert, D., Houk, K.N. & Mayo, S.L. (2012)
Iterative approach to computational enzyme design. Proc Natl Acad Sci USA 109, 3790–3795

Khersonsky, O., Röthlisberger, D., Wollacott, A.M., Murphy, P., Dym, O., Albeck, S., Kiss, G., Houk, K.N., Baker, D. & Tawfik, D.S. (2011)
Optimization of the in-silico-designed kemp eliminase KE70 by computational design and directed evolution. J Mol Biol 407, 391–412

Röthlisberger, D., Khersonsky, O., Wollacott, A.M., Jiang, L., DeChancie, J., Betker, J., Gallaher, J.L., Althoff, E.A., Zanghellini, A., Dym, O., Albeck, S., Houk, K.N., Tawfik, D.S. & Baker, D. (2008)
Kemp elimination catalysts by computational enzyme design. Nature 453, 190–195



Designing Highly Mutated Sequences

Privett, H.K., Kiss, G., Lee, T.M., Blomberg, R., Chica, R.A., Thomas, L.M., Hilvert, D., Houk, K.N. & Mayo, S.L. (2012)
Iterative approach to computational enzyme design. Proc Natl Acad Sci USA 109, 3790–3795

Siegel, J.B., Zanghellini, A., Lovick, H.M., Kiss, G., Lambert, A.R., St Clair, J.L., Gallaher, J.L., Hilvert, D., Gelb, M.H., Stoddard, B.L., Houk, K.N., Michael, F.E. & Baker, D. (2010)
Computational design of an enzyme catalyst for a stereoselective bimolecular Diels-Alder reaction. Science 329, 309–313

Jiang, L., Althoff, E.A., Clemente, F.R., Doyle, L., Röthlisberger, D., Zanghellini, A., Gallaher, J.L., Betker, J.L., Tanaka, F., Barbas, C.F., Hilvert, D., Houk, K.N., Stoddard, B.L. & Baker, D. (2008)
De novo computational design of retro-aldol enzymes. Science 319, 1387–1391

Murphy, P.M., Bolduc, J.M., Gallaher, J.L., Stoddard, B.L. & Baker, D. (2009)
Alteration of enzyme specificity by computational loop remodeling and design. PNAS 106, 9215–9220

Ashworth, J., Havranek, J.J., Duarte, C.M., Sussman, D., Monnat, R.J., Stoddard, B.L. & Baker, D. (2006)
Computational redesign of endonuclease DNA binding and cleavage specificity. Nature 441, 656–659

Jha, R.K., Leaver-Fay, A., Yin, S., Wu, Y., Butterfoss, G.L., Szyperski, T., Dokholyan, N.V. & Kuhlman, B. (2010)
Computational design of a PAK1 binding protein. J Mol Biol 400, 257–270

Huang, P.-S., Love, J.J. & Mayo, S.L. (2007)
A de novo designed protein protein interface. Protein Sci 16, 2770–2774