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Protein Evolution is Potentially Governed by Protein Stability: Directed Evolution of an Esterase from the Hyperthermophilic Archaeon Sulfolobus tokodaii

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Abstract

The study of evolution is important to understand biological phenomena. During evolutionary processes, genetic changes confer amino acid substitutions in proteins, resulting in new or improved functions. Unfortunately, most mutations destabilize proteins. Thus, protein stability is a significant factor in evolution; however, its role remains unclear. Here, we simply and directly explored the association between protein activity and stability in random mutant libraries to elucidate the role of protein stability in evolutionary processes. In the first random mutation of an esterase from Sulfolobus tokodaii, approximately 20% of the variants displayed higher activity than wild-type protein (i.e., 20% evolvability). During evolutionary processes, the evolvability depended on the stability of template proteins, indicating that protein evolution is potentially governed by protein stability. Furthermore, decreased activity could be recovered during evolution by maintaining the stability of variants. The results suggest that protein sequence space for its evolution is able to expand during nearly neutral evolution where mutations are slightly deleterious for activity but rarely fatal for stability. Molecular evolution is a crucial phenomenon that has continued since the birth of life on earth, and mechanism underlying it is simple; therefore, this could be demonstrated by our simple experiments. These findings also can be applied to protein engineering.

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References

  • Aharoni A, Gaidukov L, Khersonsky O, Gould SM, Roodveldt C, Tawfik DS (2005) The ‘evolvability’ of promiscuous protein functions. Nat Genet 37:73–76

    Article  PubMed  CAS  Google Scholar 

  • Akashi H, Osada N, Ohta T (2012) Weak selection and protein evolution. Genet 192:15–31

    Article  CAS  Google Scholar 

  • Angkawidjaja C, Koga Y, Takano K, Kanaya S (2012) Structure and stability of a thermostable carboxylesterase from the thermoacidophilic archaeon Sulfolobus tokodaii. FEBS J 279:3071–3084

    Article  PubMed  CAS  Google Scholar 

  • Ashenberg O, Gong LI, Bloom JD (2013) Mutational effects on stability are largely conserved during protein evolution. Proc Natl Acad Sci USA 110:21071–21076

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bastolla U, Dehouck Y, Echave J (2017) What evolution tells us about protein physics, and protein physics tells us about evolution. Curr Opin Struct Biol 42:59–66

    Article  PubMed  CAS  Google Scholar 

  • Berezovsky IN, Shakhnovich EI (2005) Physics and evolution of thermophilic adaptation. Proc Natl Acad Sci USA 102:12742–12747

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bershtein S, Segal M, Bekerman R, Tokuriki N, Tawfik DS (2006) Robustness–epistasis link shapes the fitness landscape of a randomly drifting protein. Nature 444(7121):929

    Article  PubMed  CAS  Google Scholar 

  • Bershtein S, Goldin K, Tawfik DS (2008) Intense neutral drifts yield robust and evolvable consensus proteins. J Mol Biol 379:1029–1044

    Article  PubMed  CAS  Google Scholar 

  • Bershtein S, Serohijos AW, Shakhnovich EI (2017) Bridging the physical scales in evolutionary biology: from protein sequence space to fitness of organisms and populations. Curr Opin Struct Biol 42:31–40

    Article  PubMed  CAS  Google Scholar 

  • Blaber M, Zhang XJ, Matthews BW (1993) Structural basis of amino acid alpha helix propensity. Science 260:1637–1640

    Article  PubMed  CAS  Google Scholar 

  • Bloom JD, Labthavikul ST, Otey CR, Arnold FH (2006) Protein stability promotes evolvability. Proc Natl Acad Sci USA 103:5869–5874

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Bloom JD, Raval A, Wilke CO (2007) Thermodynamics of neutral protein evolution. Genet 175:255–266

    Article  CAS  Google Scholar 

  • Bogumil D, Dagan T (2012) Cumulative impact of chaperone-mediated folding on genome evolution. Biochemistry 51:9941–9953

    Article  PubMed  CAS  Google Scholar 

  • Caetano-Anollés G, Mittenthal J (2010) Exploring the interplay of stability and function in protein evolution. Bioessays 32:655–658

    Article  PubMed  CAS  Google Scholar 

  • Carneiro M, Hartl DL (2010) Adaptive landscapes and protein evolution. Proc Natl Acad Sci USA 107:1747–1751

    Article  PubMed  PubMed Central  Google Scholar 

  • Darwin CR (1859) On the origin of species by means of natural selection, or the preservation of favoured races in the struggle for life. John Murray, London

    Book  Google Scholar 

  • DePristo MA, Weinreich DM, Hartl DL (2005) Missense meanderings in sequence space: a biophysical view of protein evolution. Nat Rev Genet 6:678–687

    Article  PubMed  CAS  Google Scholar 

  • Draghi JA, Parsons TL, Wagner GP, Plotkin JB (2010) Mutational robustness can facilitate adaptation. Nature 463:353–355

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Elena SF, Sanjuán R (2008) The effect of genetic robustness on evolvability in digital organisms. BMC Evol Biol 8:284

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Funahashi J, Takano K, Yamagata Y, Yutani K (1999) Contribution of amino acid substitutions at two different interior positions to the conformational stability of human lysozyme. Protein Eng 12:841–850

    Article  PubMed  CAS  Google Scholar 

  • Godoy-Ruiz R, Perez-Jimenez R, Ibarra-Molero B, Sanchez-Ruiz JM (2004) Relation between protein stability, evolution and structure, as probed by carboxylic acid mutations. J Mol Biol 336:313–318

    Article  PubMed  CAS  Google Scholar 

  • Gong LI, Suchard MA, Bloom JD (2013) Stability-mediated epistasis constrains the evolution of an influenza protein. eLife 2:e00631

    Article  PubMed  PubMed Central  Google Scholar 

  • Kaltenbach M, Tokuriki N (2014) Dynamics and constraints of enzyme evolution. J Exp Zool B 322:468–487

    Article  CAS  Google Scholar 

  • Kimura M (1968) Evolutionary rate at the molecular level. Nature 217:624–626

    Article  PubMed  CAS  Google Scholar 

  • Mack KL, Shorter J (2016) Engineering and evolution of molecular chaperones and protein disaggregases with enhanced activity. Front Mol Biosci 3:8

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Magliery TJ, Lavinder JJ, Sullivan BJ (2011) Protein stability by number: high-throughput and statistical approaches to one of protein science’s most difficult problems. Curr Opin Chem Biol 15:443–451

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Matthews BW, Nicholson H, Becktel WJ (1987) Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding. Proc Natl Acad Sci USA 84:6663–6667

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Miton CM, Tokuriki N (2016) How mutational epistasis impairs predictability in protein evolution and design. Protein Sci 25:1260–1272

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Mizuguchi K, Sele M, Cubellis MV (2007) Environment specific substitution tables for thermophilic proteins. BMC Bioinform 8:S15

    Article  CAS  Google Scholar 

  • Mukaiyama A, Haruki M, Ota M, Koga Y, Takano K, Kanaya S (2006) A hyperthermophilic protein acquires function at the cost of stability. Biochemistry 45:12673–12679

    Article  PubMed  CAS  Google Scholar 

  • Nelson KE et al (1999) Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima. Nature 399:323–329

    Article  PubMed  CAS  Google Scholar 

  • Ohta T (1973) Slightly deleterious mutant substitutions in evolution. Nature 246:96–98

    Article  PubMed  CAS  Google Scholar 

  • Ohta T (1992) The nearly neutral theory of molecular evolution. Annu Rev Ecol Syst 23:263–286

    Article  Google Scholar 

  • Okada J, Okamoto T, Mukaiyama A, Tadokoro T, You DJ, Chon H, Koga Y, Takano K, Kanaya S (2010) Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins. BMC Evol Biol 10:207

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Philip AF, Kumauchi M, Hoff WD (2010) Robustness and evolvability in the functional anatomy of a PER-ARNT-SIM (PAS) domain. Proc Natl Acad Sci USA 107:17986–17991

    Article  PubMed  PubMed Central  Google Scholar 

  • Romero PA, Arnold FH (2009) Exploring protein fitness landscapes by directed evolution. Nat Rev Mol Cell Biol 10:866–876

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Serrano L, Neira JL, Sancho J, Fersht AR (1992) Effect of alanine versus glycine in alpha-helices on protein stability. Nature 356:453–455

    Article  PubMed  CAS  Google Scholar 

  • Sikosek T, Chen HS (2014) Biophysics of protein evolution and evolutionary protein biophysics. J R Soc Interface 11:20140419

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Socha RD, Tokuriki N (2013) Modulating protein stability—directed evolution strategies for improved protein function. FEBS J 280:5582–5595

    Article  PubMed  CAS  Google Scholar 

  • Son SY, Ma J, Kondou Y, Yoshimura M, Yamashita E, Tsukihara T (2008) Structure of human monoamine oxidase A at 2.2-Å resolution: the control of opening the entry for substrates/inhibitors. Proc Natl Acad Sci USA 105:5739–5744

    Article  PubMed  PubMed Central  Google Scholar 

  • Starr TN, Thornton JW (2016) Epistasis in protein evolution. Protein Sci 25:1204–1218

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Steinberg B, Ostermeier M (2016a) Shifting fitness and epistatic landscapes reflect trade-offs along an evolutionary pathway. J Mol Biol 428:2730–2743

    Article  PubMed  CAS  Google Scholar 

  • Steinberg B, Ostermeier M (2016b) Environmental changes bridge evolutionary valleys. Sci Adv 2:e1500921

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Suzuki Y, Miyamoto K, Ohta H (2004) A novel thermostable esterase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. FEMS Microbiol Lett 236:97–102

    Article  PubMed  CAS  Google Scholar 

  • Takano K, Ogasahara K, Kaneda H, Yamagata Y, Fujii S, Kanaya E, Kikuchi M, Oobatake M, Yutani K (1995) Contribution of hydrophobic residues to the stability of human lysozyme: calorimetric studies and X-ray structural analysis of the five isoleucine to valine mutants. J Mol Biol 254:62–76

    Article  PubMed  CAS  Google Scholar 

  • Takano K, Yamagata Y, Kubota M, Funahashi J, Fujii S, Yutani K (1999) Contribution of hydrogen bonds to the conformational stability of human lysozyme: calorimetry and X-ray analysis of six Ser → Ala mutants. Biochemistry 38:6623–6629

    Article  PubMed  CAS  Google Scholar 

  • Takano K, Tsuchimori K, Yamagata Y, Yutani K (2000) Contribution of salt bridges near the surface of a protein to the conformational stability. Biochemistry 39:12375–12381

    Article  PubMed  CAS  Google Scholar 

  • Takano K, Scholtz JM, Sacchettini JC, Pace CN (2003) The contribution of polar group burial to protein stability is strongly context-dependent. J Biol Chem 278:31790–31795

    Article  PubMed  CAS  Google Scholar 

  • Takano K, Aoi A, Koga Y, Kanaya S (2013) Evolvability of thermophilic proteins from Archaea and Bacteria. Biochemistry 52:4774–4780

    Article  PubMed  CAS  Google Scholar 

  • Tokuriki N, Tawfik DS (2009a) Stability effects of mutations and protein evolvability. Curr Opin Struct Biol 19:596–604

    Article  PubMed  CAS  Google Scholar 

  • Tokuriki N, Tawfik DS (2009b) Chaperonin overexpression promotes genetic variation and enzyme evolution. Nature 459:668–673

    Article  PubMed  CAS  Google Scholar 

  • Tokuriki N, Stricher F, Schymkowitz J, Serrano L, Tawfik DS (2007) The stability effects of protein mutations appear to be universally distributed. J Mol Biol 369:1318–1332

    Article  PubMed  CAS  Google Scholar 

  • Tokuriki N, Stricher F, Serrano L, Tawfik DS (2008) How protein stability and new functions trade off. PLoS Comput Biol 4:e1000002

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Wang X, Minasov G, Shoichet BK (2002) Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs. J Mol Biol 320:85–95

    Article  PubMed  CAS  Google Scholar 

  • Worth CL, Gong S, Blundell TL (2009) Structural and functional constraints in the evolution of protein families. Nat Rev Mol Cell Biol 10:709–720

    Article  PubMed  CAS  Google Scholar 

  • Wyganowski KT, Kaltenbach M, Tokuriki N (2013) GroEL/ES buffering and compensatory mutations promote protein evolution by stabilizing folding intermediates. J Mol Biol 425:3403–3414

    Article  PubMed  CAS  Google Scholar 

  • Yang G, Hong N, Baier F, Jackson CJ, Tokuriki N (2016) Conformational tinkering drives evolution of a promiscuous activity through indirect mutational effects. Biochemistry 55:4583–4593

    Article  PubMed  CAS  Google Scholar 

  • Zeldovich KB, Chen P, Shakhnovich EI (2007a) Protein stability imposes limits on organism complexity and speed of molecular evolution. Proc Natl Acad Sci USA 104:16152–16157

    Article  PubMed  PubMed Central  Google Scholar 

  • Zeldovich KB, Chen P, Shakhnovich BE, Shakhnovich EI (2007b) A first-principles model of early evolution: emergence of gene families, species, and preferred protein folds. PLoS Comput Biol 3:e139

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

This work was supported in part by grants from the Japan Society for the Promotion of Science to KT (KAKENHI Nos. 25440194 and 17K07368), by a Research Encouragement Project from the Academic Promotion Fund of Kyoto Prefectural University to RK (2016), and by a Young Researcher Development Support Project from the Kyoto Prefectural Public University Corporation to RK (2017).

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KT conceived and supervised the experiments. RK performed the experiments. RK and KT wrote the paper. SS helped in interpretation of data and discussion of results. All the authors have read and approved the final manuscript.

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Correspondence to Kazufumi Takano.

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The authors declare no competing financial interest.

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Kurahashi, R., Sano, S. & Takano, K. Protein Evolution is Potentially Governed by Protein Stability: Directed Evolution of an Esterase from the Hyperthermophilic Archaeon Sulfolobus tokodaii. J Mol Evol 86, 283–292 (2018). https://doi.org/10.1007/s00239-018-9843-y

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