Skip to main content
SpringerLink
Log in

Denaturation of an extremely stable hyperthermophilic protein occurs via a dimeric intermediate

  • Original Paper
  • Published:
Extremophiles Aims and scope Submit manuscript

Abstract

To elucidate determinants of thermostability and folding pathways of the intrinsically stable proteins from extremophilic organisms, we are studying β-glucosidase from Pyrococcus furiosus. Using fluorescence and circular dichroism spectroscopy, we have characterized the thermostability of β-glucosidase at 90°C, the lowest temperature where full unfolding is achieved with urea. The chemical denaturation profile reveals that this homotetrameric protein unfolds at 90°C with an overall ΔG° of ∼ 20 kcal mol−1. The high temperatures needed to chemically denature P. furiosus β-glucosidase and the large ΔG° of unfolding at high temperatures shows this to be one of the most stable proteins yet characterized. Unfolding proceeds via a three-state pathway that includes a stable intermediate species. Stability of the native and intermediate forms is concentration dependent, and we have identified a dimeric assembly intermediate using high temperature native gel electrophoresis. Based on this data, we have developed a model for the denaturation of β-glucosidase in which the tetramer dissociates to partially folded dimers, followed by the coupled dissociation and denaturation of the dimers to unfolded monomers. The extremely high stability is thus derived from a combination of oligomeric interactions and subunit folding.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

GdnHCl:

Guanidine hydrochloride

CD:

Circular dichroism

References

  • Adams MW, Kelly RM (1998) Finding and using hyperthermophilic enzymes. Trends Biotechnol 16:329–332

    Article  PubMed  CAS  Google Scholar 

  • Barry JK, Matthews KS (1999) Thermodynamic analysis of unfolding and dissociation in lactose repressor protein. Biochemistry 38:6520–6528

    Article  PubMed  CAS  Google Scholar 

  • Bauer MW, Bylina EJ, Swanson RV, Kelly RM (1996) Comparison of a beta-glucosidase and a beta-mannosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Purification, characterization, gene cloning, and sequence analysis. J Biol Chem 271: 23749–23755

    Article  PubMed  CAS  Google Scholar 

  • Bauer MW, Driskill LE, Kelly RM (1998) Glycosyl hydrolases from hyperthermophilic microorganisms. Curr Opin Biotechnol 9:141–145

    Article  PubMed  CAS  Google Scholar 

  • Benton CB, King J, Clark PL (2002) Characterization of the protrimer intermediate in the folding pathway of the interdigitated beta-helix tailspike protein. Biochemistry 41:5093–5103

    Article  PubMed  CAS  Google Scholar 

  • Catanzano F, Graziano G, De Paola B, Barone G, D’Auria S, Rossi M, Nucci R (1998) Guanidine-induced denaturation of beta-glycosidase from Sulfolobus solfataricus expressed in Escherichia coli. Biochemistry 37:14484–14490

    Article  PubMed  CAS  Google Scholar 

  • Chen H, Chu Z, Zhang Y, Yang S (2006) Over-expression and characterization of the recombinant small heat shock protein from Pyrococcus furiosus. Biotechnol Lett 28:1089–1094

    Article  PubMed  CAS  Google Scholar 

  • D’Amico S, Marx JC, Gerday C, Feller G (2003) Activity–stability relationships in extremophilic enzymes. J Biol Chem 278: 7891–7896

    Article  PubMed  CAS  Google Scholar 

  • Demirjian DC, Moris-Varas F, Cassidy CS (2001) Enzymes from extremophiles. Curr Opin Chem Biol 5:144–151

    Article  PubMed  CAS  Google Scholar 

  • Eftink MR (1994) The use of fluorescence methods to monitor unfolding transitions of proteins. Biophys J 66:482–501

    PubMed  CAS  Google Scholar 

  • Feller G, d’Amico D, Gerday C (1999) Thermodynamic stability of a cold-active alpha-amylase from the Antarctic bacterium Alteromonas haloplanctis. Biochemistry 38:4613–4619

    Article  PubMed  CAS  Google Scholar 

  • Ferguson KA (1964) Starch-gel electrophoresis–application to the classification of pituitary proteins and polypeptides. Metabolism 13 (Suppl):985–1002

    Google Scholar 

  • Fiala G, Stetter Karl O (1986) Pyrococcus furiosus sp. nov. represents a novel genus of marine heterotrophic archaebacteria growing optimally at 100°C. Arch Microbiol 145:56–61

    Article  CAS  Google Scholar 

  • Gerding JJ, Koppers A, Hagel P, Bloemendal H (1971) Cyanate formation in solutions of urea. II. Effect of urea on the eye lens protein-crystallin. Biochim Biophys Acta 243:375–379

    PubMed  CAS  Google Scholar 

  • Ghosh M, Mandal DK (2001) Analysis of equilibrium dissociation and unfolding in denaturants of soybean agglutinin and two of its derivatives. Int J Biol Macromol 29:273–280

    Article  PubMed  CAS  Google Scholar 

  • Grimsley JK, Scholtz JM, Pace CN, Wild JR (1997) Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric intermediate. Biochemistry 36:14366–14374

    Article  PubMed  CAS  Google Scholar 

  • Haeberle JR (1997) High-temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis. Biotechniques 23:638–640

    PubMed  CAS  Google Scholar 

  • Hames D (1990) One-dimensional polyacrylamide gel electrophoresis. In: Rickwood BD, Ha D (eds) Gel electrophoresis of proteins: a practical approach. Oxford University Press, Oxford, pp 1–147

    Google Scholar 

  • Hobart SA, Meinhold DW, Osuna R, Colon W (2002) From two-state to three-state: the effect of the P61A mutation on the dynamics and stability of the factor for inversion stimulation results in an altered equilibrium denaturation mechanism. Biochemistry 41:13744–13754

    Article  PubMed  CAS  Google Scholar 

  • Kaper T, Lebbink JH, Pouwels J, Kopp J, Schulz GE, van der Oost J, de Vos WM (2000) Comparative structural analysis and substrate specificity engineering of the hyperthermostable beta-glucosidase CelB from Pyrococcus furiosus. Biochemistry 39:4963–4970

    Article  PubMed  CAS  Google Scholar 

  • Kengen SW, Luesink EJ, Stams AJ, Zehnder AJ (1993) Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus. Eur J Biochem 213:305–312

    Article  PubMed  CAS  Google Scholar 

  • Koutsopoulos S, van der Oost J, Norde W (2005) Conformational studies of a hyperthermostable enzyme. FEBS J 272:5484–5496

    Article  PubMed  CAS  Google Scholar 

  • Ladokhin AS, Jayasinghe, Sajith, White Stephen H. (2000) How to measure and analyze tryptophan fluorescence in membranes properly, and why bother? Anal Biochem 285:235–245

    Article  PubMed  CAS  Google Scholar 

  • Machielsen R, van der Oost J (2006) Production and characterization of a thermostable l-threonine dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus. FEBS J 273:2722–2729

    Article  PubMed  CAS  Google Scholar 

  • Mamat B, Roth A, Grimm C, Ermler U, Tziatzios C, Schubert D, Thauer RK, Shima S (2002) Crystal structures and enzymatic properties of three formyltransferases from archaea: environmental adaptation and evolutionary relationship. Protein Sci 11:2168–2178

    Article  PubMed  CAS  Google Scholar 

  • Marquardt DW (1963) An algorithm for least squares estimation of non-linear parameters. J Appl Math 11:431–441

    Google Scholar 

  • Ogasahara K, Ishida M, Yutani K (2003) Stimulated interaction between and subunits of tryptophan synthase from hyperthermophile enhances its thermal stability. J Biol Chem 278:8922–8928

    Article  PubMed  CAS  Google Scholar 

  • Pace CN (1986) Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol 131:266–280

    Article  PubMed  CAS  Google Scholar 

  • Pouwels J, Moracci M, Cobucci-Ponzano B, Perugino G, van der Oost J, Kaper T, Lebbink JH, de Vos WM, Ciaramella M, Rossi M (2000) Activity and stability of hyperthermophilic enzymes: a comparative study on two archaeal beta-glycosidases. Extremophiles 4:157–164

    Article  PubMed  CAS  Google Scholar 

  • Smith JD, Robinson AS (2002) Overexpression of an archaeal protein in yeast: secretion bottleneck at the ER. Biotechnol Bioeng 79:713–723

    Article  PubMed  CAS  Google Scholar 

  • Tatur J, Hagedoorn PL, Overeijnder ML, Hagen WR (2006) A highly thermostable ferritin from the hyperthermophilic archaeal anaerobe Pyrococcus furiosus. Extremophiles 10:139–148

    Article  PubMed  CAS  Google Scholar 

  • Timm DE, Neet KE (1992) Equilibrium denaturation studies of mouse beta-nerve growth factor. Protein Sci 1:236–244

    Article  PubMed  CAS  Google Scholar 

  • Voorhorst WG, Eggen RI, Luesink EJ, de Vos WM (1995) Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli. J Bacteriol 177:7105–7111

    PubMed  CAS  Google Scholar 

  • Wallecha A, Mishra S (2003) Purification and characterization of two beta-glucosidases from a thermo-tolerant yeast Pichia etchellsii. Biochim Biophys Acta 1649:74–84

    PubMed  CAS  Google Scholar 

  • Wood T, Bhat KM (1988) Biomass. Part A. Cellulose and hemicellulose. In: Wood WA, Kellogg ST (eds) Methods in enzymology. Academic, San Diego, pp 87–112

    Google Scholar 

Download references

Acknowledgments

The authors thank Dr. Robert M. Kelly (NC State University) for the generous gift of the β-glucosidase gene. We also thank Dr. Jason Smith, Dr. Tzvetana Lazarova, and Dr. Yu-Sung Wu for experimental assistance and helpful discussions, and Dr. Babatunde Ogunnaike and Claudio Gelmi for discussion of statistics and error analysis. This study was supported by NIH T32 GM 08550-09 (SLP), NSF BES99-84312, and the University of Delaware Research Foundation.

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, University of Delaware, Newark, DE, 19716, USA

    Sara Lawrence Powers & Anne Skaja Robinson

  2. Department of Chemistry and Biochemistry, University of Delaware, Newark, DE, 19716, USA

    Clifford R. Robinson

Authors
  1. Sara Lawrence Powers
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Clifford R. Robinson
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anne Skaja Robinson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Anne Skaja Robinson.

Additional information

Communicated by A. Driessen.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Powers, S.L., Robinson, C.R. & Robinson, A.S. Denaturation of an extremely stable hyperthermophilic protein occurs via a dimeric intermediate. Extremophiles 11, 179–189 (2007). https://doi.org/10.1007/s00792-006-0030-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00792-006-0030-5

Keywords

Search

Navigation