There is great interest in increasing proteins' stability to enhance their utility as biocatalysts, therapeutics, diagnostics and nanomaterials. Directed evolution is a powerful, but experimentally strenuous approach. Computational methods offer attractive alternatives. However, due to the limited reliability of predictions and potentially antagonistic effects of substitutions, only single-point mutations are usually predicted in silico, experimentally verified and then recombined in multiple-point mutants. Thus, substantial screening is still required. Here we present FireProt, a robust computational strategy for predicting highly stable multiple-point mutants that combines energy- and evolution-based approaches with smart filtering to identify additive stabilizing mutations. FireProt's reliability and applicability was demonstrated by validating its predictions against 656 mutations from the ProTherm database. We demonstrate that thermostability of the model enzymes haloalkane dehalogenase DhaA and γ-hexachlorocyclohexane dehydrochlorinase LinA can be substantially increased (ΔTm = 24°C and 21°C) by constructing and characterizing only a handful of multiple-point mutants. FireProt can be applied to any protein for which a tertiary structure and homologous sequences are available, and will facilitate the rapid development of robust proteins for biomedical and biotechnological applications.

Department of Biochemistry University of Washington Seattle Washington United States of America

Department of Chemistry and Chemical Biology Rutgers University Piscataway New Jersey United States of America

Loschmidt Laboratories Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX Masaryk University Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX Masaryk University Brno Czech Republic; International Clinical Research Center St Anne's University Hospital Brno Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX Masaryk University Brno Czech Republic; International Clinical Research Center St Anne's University Hospital Brno Brno Czech Republic; Department of Information Systems Faculty of Information Technology Brno University of Technology Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOX Masaryk University Brno Czech Republic; International Clinical Research Center St Anne's University Hospital Brno Brno Czech Republic; Enantis Ltd Brno Czech Republic

Zobrazit více v PubMed

Khoury GA, Smadbeck J, Kieslich CA, Floudas CA. (2014) Protein folding and de novo protein design for biotechnological applications. Trends Biotechnol. 32: 99–109. 10.1016/j.tibtech.2013.10.008 . PubMed DOI PMC

Hartmann M, Roeraade J, Stoll D, Templin MF, Joos TO. (2009) Protein microarrays for diagnostic assays. Anal Bioanal Chem. 393: 1407–1416. 10.1007/s00216-008-2379-z . PubMed DOI

Schmid A, Hollmann F, Park JB, Buhler B. (2002) The use of enzymes in the chemical industry in Europe. Curr Opin Biotechnol. 13: 359–366. 10.1016/s0958-1669(02)00336-1 . PubMed DOI

Polizzi KM, Bommarius AS, Broering JM, Chaparro-Riggers JF. (2007) Stability of biocatalysts. Curr Opin Chem Biol. 11: 220–225. 10.1016/j.cbpa.2007.01.685 . PubMed DOI

Ferdjani S, Ionita M, Roy B, Dion M, Djeghaba Z, Rabiller C, et al. (2011) Correlation between thermostability and stability of glycosidases in ionic liquid. Biotechnol Lett. 33: 1215–1219. 10.1007/s10529-011-0560-5 . PubMed DOI

Gao D, Narasimhan DL, Macdonald J, Brim R, Ko M-C, Landry DW, et al. (2009) Thermostable variants of cocaine esterase for long-time protection against cocaine toxicity. Mol Pharmacol. 75: 318–323. 10.1124/mol.108.049486 . PubMed DOI PMC

Wijma HJ, Floor RJ, Janssen DB. (2013) Structure- and sequence-analysis inspired engineering of proteins for enhanced thermostability. Curr Opin Struct Biol. 23: 588–594. 10.1016/j.sbi.2013.04.008 . PubMed DOI

Bommarius AS, Paye MF. (2013) Stabilizing biocatalysts. Chem Soc Rev. 42: 6534–6565. 10.1039/c3cs60137d . PubMed DOI

Bloom JD, Labthavikul ST, Otey CR, Arnold FH. (2006) Protein stability promotes evolvability. Proc Natl Acad Sci U S A. 103: 5869–5874. 10.1073/pnas.0510098103 . PubMed DOI PMC

Gumulya Y, Reetz MT. (2011) Enhancing the thermal robustness of an enzyme by directed evolution: least favorable starting points and inferior mutants can map superior evolutionary pathways. ChemBioChem. 12: 2502–2510. 10.1002/cbic.201100412 . PubMed DOI

Seitz T, Thoma R, Schoch GA, Stihle M, Benz J, D'Arcy B, et al. (2010) Enhancing the stability and solubility of the glucocorticoid receptor ligand-binding domain by high-throughput library screening. J Mol Biol. 403: 562–577. 10.1016/j.jmb.2010.08.048 . PubMed DOI

Damborsky J, Brezovsky J. (2014) Computational methods for designing and engineering biocatalysts. Curr Opin Chem Biol. 19: 8–16. 10.1016/j.cbpa.2013.12.003 PubMed DOI

Koudelakova T, Chaloupkova R, Brezovsky J, Prokop Z, Sebestova E, Hesseler M, et al. (2013) Engineering enzyme stability and resistance to an organic cosolvent by modification of residues in the access tunnel. Angew Chem Int Ed. 52: 1959–1963. 10.1002/anie.201206708 . PubMed DOI

Bosshart A, Panke S, Bechtold M. (2013) Systematic optimization of interface interactions increases the thermostability of a multimeric enzyme. Angew Chem Int Ed. 52: 9673–9676. 10.1002/anie.201304141 . PubMed DOI

Wijma HJ, Floor RJ, Jekel PA, Baker D, Siewert MJ, Janssen DB. (2014) Computationally designed libraries for rapid enzyme stabilization. Protein Eng Des Sel. 27: 49–58. 10.1093/protein/gzt061 PubMed DOI PMC

Komor RS, Romero PA, Xie CB, Arnold FH. (2012) Highly thermostable fungal cellobiohydrolase I (Cel7A) engineered using predictive methods. Protein Eng Des Sel. 25: 827–833. 10.1093/protein/gzs058 . PubMed DOI

Reetz MT, Soni P, Acevedo JP, Sanchis J. (2009) Creation of an amino acid network of structurally coupled residues in the directed evolution of a thermostable enzyme. Angew Chem Int Ed. 48: 8268–8272. 10.1002/anie.200904209 . PubMed DOI

Kumar MDS, Bava KA, Gromiha MM, Prabakaran P, Kitajima K, Uedaira H, et al. (2006) ProTherm and ProNIT: thermodynamic databases for proteins and protein-nucleic acid interactions. Nucleic Acids Res. 34: D204–D206. 10.1093/nar/gkj103 . PubMed DOI PMC

Khatun J, Khare SD, Dokholyan NV. (2004) Can contact potentials reliably predict stability of proteins? J Mol Biol. 336: 1223–1238. 10.1016/j.jmb.2004.01.002 . PubMed DOI

Guerois R, Nielsen JE, Serrano L. (2002) Predicting changes in the stability of proteins and protein complexes: A study of more than 1000 mutations. J Mol Biol. 320: 369–387. 10.1016/s0022-2836(02)00442-4 . PubMed DOI

Kellogg EH, Leaver-Fay A, Baker D. (2011) Role of conformational sampling in computing mutation-induced changes in protein structure and stability. Protein Struct Funct Bioinformatics. 79: 830–838. 10.1002/prot.22921 . PubMed DOI PMC

Yin S, Ding F, Dokholyan NV. (2007) Eris: an automated estimator of protein stability. Nature Methods. 4: 466–467. 10.1038/nmeth0607-466 . PubMed DOI

Parthiban V, Gromiha MM, Schomburg D. (2006) CUPSAT: prediction of protein stability upon point mutations. Nucleic Acids Res. 34: W239–W242. 10.1093/nar/gkl190 . PubMed DOI PMC

Pupko T, Bell RE, Mayrose I, Glaser F, Ben-Tal N. (2002) Rate4Site: an algorithmic tool for the identification of functional regions in proteins by surface mapping of evolutionary determinants within their homologues. Bioinformatics. 18: S71–77. PubMed

Landau M, Mayrose I, Rosenberg Y, Glaser F, Martz E, Pupko T, et al. (2005) ConSurf 2005: the projection of evolutionary conservation scores of residues on protein structures. Nucleic Acids Res. 33: W299–W302. 10.1093/nar/gki370 . PubMed DOI PMC

Ashkenazy H, Erez E, Martz E, Pupko T, Ben-Tal N. (2010) ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38: W529–W533. 10.1093/nar/gkq399 . PubMed DOI PMC

Lehmann M, Pasamontes L, Lassen SF, Wyss M. (2000) The consensus concept for thermostability engineering of proteins. Biochim Biophys Acta, Protein Structure M. 1543: 408–415. 10.1016/s0167-4838(00)00238-7 . PubMed DOI

Gray KA, Richardson TH, Kretz K, Short JM, Bartnek F, Knowles R, et al. (2001) Rapid evolution of reversible denaturation and elevated melting temperature in a microbial haloalkane dehalogenase. Adv Synth Catal. 343: 607–616.

Kuipers RKP, Joosten H-J, Verwiel E, Paans S, Akerboom J, van der Oost J, et al. (2009) Correlated mutation analyses on super-family alignments reveal functionally important residues. Protein Struct Funct Bioinformatics. 76: 608–616. 10.1002/prot.22374 . PubMed DOI

Koudelakova T, Bidmanova S, Dvorak P, Pavelka A, Chaloupkova R, Prokop Z, et al. (2013) Haloalkane dehalogenases: Biotechnological applications. Biotechnol J. 8: 32–45. 10.1002/biot.201100486 . PubMed DOI

Borgo B, Havranek JJ. (2012) Automated selection of stabilizing mutations in designed and natural proteins. Proc Natl Acad Sci U S A. 109: 1494–1499. 10.1073/pnas.1115172109 . PubMed DOI PMC

Diaz JE, Lin C-S, Kunishiro K, Feld BK, Avrantinis SK, Bronson J, et al. (2011) Computational design and selections for an engineered, thermostable terpene synthase. Protein Sci. 20: 1597–1606. 10.1002/pro.691 . PubMed DOI PMC

Floor RJ, Wijma HJ, Colpa DI, Ramos-Silva A, Jekel PA, Szymanski W, et al. (2014) Computational library design for increasing haloalkane dehalogenase stability. ChemBioChem. 15: 1659–1671. 10.1002/cbic.201402128 . PubMed DOI

Pavelka A, Chovancova E, Damborsky J. (2009) HotSpot Wizard: a web server for identification of hot spots in protein engineering. Nucleic Acids Res. 37: W376–W383. 10.1093/nar/gkp410 . PubMed DOI PMC

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389–3402. PubMed PMC

Sayers EW, Barrett T, Benson DA, Bryant SH, Canese K, Chetvernin V, et al. (2009) Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 37: D5–D15. 10.1093/nar/gkn741 . PubMed DOI PMC

Li WZ, Godzik A. (2006) Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 22: 1658–1659. 10.1093/bioinformatics/btl158 . PubMed DOI

Frickey T, Lupas A. (2004) CLANS: a Java application for visualizing protein families based on pairwise similarity. Bioinformatics. 20: 3702–3704. . PubMed

Edgar RC. (2004) MUSCLE: a multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics. 5: 1–19. 10.1186/1471-2105-5-113 . PubMed DOI PMC

Mika S, Rost B. (2003) UniqueProt: creating representative protein sequence sets. Nucleic Acids Res. 31: 3789–3791. 10.1093/nar/gkg620 . PubMed DOI PMC

Whelan S, Goldman N. (2001) A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach. Mol Biol Evol. 18: 691–699. . PubMed

Murzin AG, Brenner SE, Hubbard T, Chothia C. (1995) SCOP: a structural classification of proteins database for the investigation of sequences and structures. J Mol Biol. 247: 536–540. 10.1016/s0022-2836(05)80134-2 . PubMed DOI

Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, et al. (2000) The Protein Data Bank. Nucleic Acids Res. 28: 235–242. PubMed PMC

The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC.

Costantini S, Colonna G, Facchiano AM. (2008) ESBRI: A web server for evaluating salt bridges in proteins. Bioinformation. 3: 137–138. PubMed PMC

Tina KG, Bhadra R, Srinivasan N. (2007) PIC: Protein Interactions Calculator. Nucleic Acids Res. 35: W473–W476. 10.1093/nar/gkm423 . PubMed DOI PMC

Pavlova M, Klvana M, Prokop Z, Chaloupkova R, Banas P, Otyepka M, et al. (2009) Redesigning dehalogenase access tunnels as a strategy for degrading an anthropogenic substrate. Nat Chem Biol. 5: 727–733. 10.1038/nchembio.205 PubMed DOI

Okai M, Kubota K, Fukuda M, Nagata Y, Nagata K, Tanokura M. (2009) Crystallization and preliminary X-ray analysis of gamma-hexachlorocyclohexane dehydrochlorinase LinA from Sphingobium japonicum UT26. Acta Crystallographica Section F-Structural Biology and Crystallization Communications. 65: 822–824. 10.1107/s1744309109026645 . PubMed DOI PMC

Stepankova V, Damborsky J, Chaloupkova R. (2013) Organic co-solvents affect activity, stability and enantioselectivity of haloalkane dehalogenases. Biotechnol J. 8: 719–729. 10.1002/biot.201200378 . PubMed DOI

Iwasaki I, Utsumi S, Ozawa T. (1952) New colorimetric determination of chloride using mercuric thiocyanate and ferric ion. Bull Chem Soc Jpn. 25: 226.

Kelly SM, Jess TJ, Price NC. (2005) How to study proteins by circular dichroism. Biochim Biophys Acta. 1751: 119–139. 10.1016/j.bbapap.2005.06.005 . PubMed DOI

Ladbrooke BD, Williams RM, Chapman D. (1968) Studies on lecithin-cholesterol-water interactions by differential scanning calorimetry and X-ray diffraction. Biochim Biophys Acta. 150: 333–340. 10.1016/0005-2736(68)90132-6 . PubMed DOI

Structural analysis of the stable form of fibroblast growth factor 2 - FGF2-STAB

Computer-aided engineering of stabilized fibroblast growth factor 21

FireProt 2.0: web-based platform for the fully automated design of thermostable proteins

Computer-assisted stabilization of fibroblast growth factor FGF-18

Advancing Enzyme's Stability and Catalytic Efficiency through Synergy of Force-Field Calculations, Evolutionary Analysis, and Machine Learning

Community-Wide Experimental Evaluation of the PROSS Stability-Design Method

Decoding the intricate network of molecular interactions of a hyperstable engineered biocatalyst

Fluorescent substrates for haloalkane dehalogenases: Novel probes for mechanistic studies and protein labeling

HotSpot Wizard 3.0: web server for automated design of mutations and smart libraries based on sequence input information

FireProt: web server for automated design of thermostable proteins

HotSpot Wizard 2.0: automated design of site-specific mutations and smart libraries in protein engineering