Thermodynamic and Kinetic Stability of a Large Multi-domain Enzyme from the Hyperthermophile Aeropyrum Pernix

Overview

Journal Extremophiles

Publisher Springer

Date 2010 Jan 9

PMID 20058042

Citations 1

Authors

Mikael Karlstrom

Roberta Chiaraluce

Laura Giangiacomo

Ida Helene Steen

Nils-Kare Birkeland

Rudolf Ladenstein

Valerio Consalvi

Affiliations

Soon will be listed here.

Abstract

The multi-domain enzyme isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix was studied by denaturant-induced unfolding. At pH 7.5, changes in circular dichroism ellipticity and intrinsic fluorescence showed a complex unfolding transition, whereas at pH 3.0, an apparently two-state and highly reversible unfolding occurred. Analytical ultracentrifugation revealed the dissociation from dimer to monomer at pH 3.0. The thermodynamic and kinetic stability were studied at pH 3.0 to explore the role of inter-domain interactions independently of inter-subunit interplay on the wild type and R211M, a mutant where a seven-membered inter-domain ionic network has been disrupted. The unfolding and folding transitions occurred at slightly different denaturant concentrations even after prolonged equilibration time. The difference between the folding and the unfolding profiles was decreased in the mutant R211M. The apparent Gibbs free energy decreased approximately 2 kcal/mol and the unfolding rate increased 4.3-fold in the mutant protein, corresponding to a decrease in activation free energy of unfolding of 0.86 kcal/mol. These results suggest that the inter-domain ionic network might be responsible for additional stabilization through a significant kinetic barrier in the unfolding pathway that could also explain the larger difference observed between the folding and unfolding transitions of the wild type.

Citing Articles

A Brief History of Colour, the Environmental Impact of Synthetic Dyes and Removal by Using Laccases.

Ardila-Leal L, Poutou-Pinales R, Pedroza-Rodriguez A, Quevedo-Hidalgo B Molecules. 2021; 26(13).

PMID: 34206669 PMC: 8270347. DOI: 10.3390/molecules26133813.

References

Benitez-Cardoza C, Rojo-Dominguez A, Hernandez-Arana A . Temperature-induced denaturation and renaturation of triosephosphate isomerase from Saccharomyces cerevisiae: evidence of dimerization coupled to refolding of the thermally unfolded protein. Biochemistry. 2001; 40(30):9049-58. DOI: 10.1021/bi010528w. View

Thurlkill R, Grimsley G, Scholtz J, Pace C . Hydrogen bonding markedly reduces the pK of buried carboxyl groups in proteins. J Mol Biol. 2006; 362(3):594-604. DOI: 10.1016/j.jmb.2006.07.056. View

Hagel P, Gerding J, Fieggen W, Bloemendal H . Cyanate formation in solutions of urea. I. Calculation of cyanate concentrations at different temperature and pH. Biochim Biophys Acta. 1971; 243(3):366-73. DOI: 10.1016/0005-2795(71)90003-1. View

Galani D, Fersht A, Perrett S . Folding of the yeast prion protein Ure2: kinetic evidence for folding and unfolding intermediates. J Mol Biol. 2002; 315(2):213-27. DOI: 10.1006/jmbi.2001.5234. View

Baker D, Agard D . Kinetics versus thermodynamics in protein folding. Biochemistry. 1994; 33(24):7505-9. DOI: 10.1021/bi00190a002. View

Karlstrom M, Stokke R, Steen I, Birkeland N, Ladenstein R . Isocitrate dehydrogenase from the hyperthermophile Aeropyrum pernix: X-ray structure analysis of a ternary enzyme-substrate complex and thermal stability. J Mol Biol. 2004; 345(3):559-77. DOI: 10.1016/j.jmb.2004.10.025. View

Vetriani C, Maeder D, Tolliday N, Yip K, Stillman T, Britton K . Protein thermostability above 100 degreesC: a key role for ionic interactions. Proc Natl Acad Sci U S A. 1998; 95(21):12300-5. PMC: 22826. DOI: 10.1073/pnas.95.21.12300. View

Goto Y, Calciano L, Fink A . Acid-induced folding of proteins. Proc Natl Acad Sci U S A. 1990; 87(2):573-7. PMC: 53307. DOI: 10.1073/pnas.87.2.573. View

Rumfeldt J, Stathopulos P, Chakrabarrty A, Lepock J, Meiering E . Mechanism and thermodynamics of guanidinium chloride-induced denaturation of ALS-associated mutant Cu,Zn superoxide dismutases. J Mol Biol. 2005; 355(1):106-23. DOI: 10.1016/j.jmb.2005.10.042. View

10.

Santoro M, Bolen D . Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry. 1988; 27(21):8063-8. DOI: 10.1021/bi00421a014. View

11.

Perrett S, Freeman S, Butler P, Fersht A . Equilibrium folding properties of the yeast prion protein determinant Ure2. J Mol Biol. 1999; 290(1):331-45. DOI: 10.1006/jmbi.1999.2872. View

12.

Dams T, Jaenicke R . Stability and folding of dihydrofolate reductase from the hyperthermophilic bacterium Thermotoga maritima. Biochemistry. 1999; 38(28):9169-78. DOI: 10.1021/bi990635e. View

13.

Lai Z, McCulloch J, Lashuel H, Kelly J . Guanidine hydrochloride-induced denaturation and refolding of transthyretin exhibits a marked hysteresis: equilibria with high kinetic barriers. Biochemistry. 1997; 36(33):10230-9. DOI: 10.1021/bi963195p. View

14.

Qin J, Clore G, Gronenborn A . Ionization equilibria for side-chain carboxyl groups in oxidized and reduced human thioredoxin and in the complex with its target peptide from the transcription factor NF kappa B. Biochemistry. 1996; 35(1):7-13. DOI: 10.1021/bi952299h. View

15.

DIRNHUBER P, Schutz F . The isomeric transformation of urea into ammonium cyanate in aqueous solutions. Biochem J. 1948; 42(4):628-32. PMC: 1258793. View

16.

DAmico S, Marx J, Gerday C, Feller G . Activity-stability relationships in extremophilic enzymes. J Biol Chem. 2003; 278(10):7891-6. DOI: 10.1074/jbc.M212508200. View

17.

Schuck P, Rossmanith P . Determination of the sedimentation coefficient distribution by least-squares boundary modeling. Biopolymers. 2000; 54(5):328-41. DOI: 10.1002/1097-0282(20001015)54:5<328::AID-BIP40>3.0.CO;2-P. View

18.

Luke K, Higgins C, Wittung-Stafshede P . Thermodynamic stability and folding of proteins from hyperthermophilic organisms. FEBS J. 2007; 274(16):4023-33. DOI: 10.1111/j.1742-4658.2007.05955.x. View

19.

Privalov P . Stability of proteins. Proteins which do not present a single cooperative system. Adv Protein Chem. 1982; 35:1-104. View

20.

Johnson M, Correia J, YPHANTIS D, Halvorson H . Analysis of data from the analytical ultracentrifuge by nonlinear least-squares techniques. Biophys J. 1981; 36(3):575-88. PMC: 1327647. DOI: 10.1016/S0006-3495(81)84753-4. View