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Heat-induced conformational changes of TET peptidase from crenarchaeon Desulfurococcus kamchatkensis

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Abstract

The effects of heating on the structure and stability of multimeric TET aminopeptidase (APDkam589) were studied by differential scanning calorimetry, tryptophan fluorescence quenching, and dynamic light scattering. Thermally induced structural changes in APDkam589 were found to occur in two phases: local conformational changes, which occur below 70 °C and are not associated with thermal denaturation of the protein, and global structural changes (above 70 °C) induced by irreversible thermal unfolding of the protein accompanied by its spontaneous aggregation. These results may explain the bell-shaped temperature dependence with a maximum at ~70 °C previously observed for enzymatic activity of APDkam589. Interestingly, the thermal unfolding of APDkam589 at about 81.2 °C is accompanied by a so-called blue-shift of about 10 nm—a shift of the Trp fluorescence spectrum toward shorter wavelength. From this point of view, APDkam589 is quite different from most proteins, which are characterized by a long wavelength shift of the spectrum (“red-shift”) upon denaturation. The blue-shift of the Trp fluorescence spectrum reflects the changes in the environment of Trp residues, which becomes more hydrophobic upon denaturation. The molecular structure of APDkam589 was determined by X-ray diffraction. The monomer of APDkam589 has six Trp residues, five of which are on the external surface of the dodecamer. Therefore, the blue-shift of the Trp fluorescence spectrum can be explained, at least partly, by aggregation of APDkam589, which occurs simultaneously with its thermal denaturation and probably makes the environment of these Trp residues more hydrophobic.

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Abbreviations

OD600 :

Optical density at 600 nm

MEROPS:

Database of proteolytic enzymes

MOPS:

3-(N-Morpholino)propanesulfonic acid

CD:

Circular dichroism spectroscopy

DSC:

Differential scanning calorimetry

DLS:

Dynamic light scattering

References

  • Ando S, Ishikawa K, Ishida H, Kawarabayasi Y, Kikuchi H, Kosugi Y (1999) Thermostable aminopeptidase from Pyrococcus horikoshii. FEBS Lett 447:25–28

    Article  CAS  PubMed  Google Scholar 

  • Andrade MA, Chacón P, Merelo JJ, Morán F (1993) Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network. Protein Eng 6(4):383–390

    Article  CAS  PubMed  Google Scholar 

  • Appolaire A, Rosenbaum E, Durá MA, Colombo M, Marty V, Savoye MN, Godfroy A, Schoehn G, Girard E, Gabel F, Franzetti B (2013) Pyrococcus horikoshii TET2 peptidase assembling process and associated functional regulation. J Biol Chem 288(31):22542–22554

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Borissenko L, Groll M (2005) Crystal structure of TET protease reveals complementary protein degradation pathways in prokaryotes. J Mol Biol 346:1207–1219

    Article  CAS  PubMed  Google Scholar 

  • Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein—dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Brandstetter H, Kim JS, Groll M, Huber R (2001) Crystal structure of the tricorn protease reveals a protein disassembly line. Nature 414:466–470

    Article  CAS  PubMed  Google Scholar 

  • Durá MA, Receveur-Brechot V, Andrieu JP, Ebel C, Schoehn G, Roussel A, Franzetti B (2005) Characterization of a TET-like aminopeptidase complex from the hyperthermophilic archaeon Pyrococcus horikoshii. Biochemistry 44:3477–3486

    Article  PubMed  Google Scholar 

  • Durá MA, Rosenbaum E, Larabi A, Gabel F, Vellieux FMD, Franzetti B (2009) The structural and biochemical characterizations of a novel TET peptidase complex from Pyrococcus horikoshii reveal an integrated peptide degradation system in hyperthermophilic Archaea. Mol Microbiol 72:26–40

    Article  PubMed  Google Scholar 

  • Duy C, Fitter J (2006) How aggregation and conformational scrambling of unfolded states govern fluorescence emission spectra. Biophys J 90(10):3704–3711

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Franzetti B, Schoehn G, Hernandez JF, Jaquinod M, Ruigrok RW, Zaccai G (2002) Tetrahedral aminopeptidase: a novel large protease complex from archaea. EMBO J 21:2132–2138

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Geier E, Pfeifer G, Wilm M, Lucchiari-Hartz M, Baumeister W, Eichmann K, Niedermann G (1999) A giant protease with potential to substitute for some functions of the proteasome. Science 283:978–981

    Article  CAS  PubMed  Google Scholar 

  • Groll M, Bochtler M, Brandstetter H, Clausen T, Huber R (2005) Molecular machines for protein degradation. ChemBioChem 6:222–256

    Article  CAS  PubMed  Google Scholar 

  • Holz R, Bzymek KP, Swierczek SI (2003) Co-catalytic metallopeptidases as pharmaceutical targets. Curr Opin Chem Biol 7:197–206

    Article  CAS  PubMed  Google Scholar 

  • Joshua-Tor L, Xu HE, Johnston SA, Rees DC (1995) Crystal structure of a conserved protease that binds DNA: the bleomycin hydrolase, Gal6. Science 269:945–950

    Article  CAS  PubMed  Google Scholar 

  • Khanova HA, Markossian KA, Kurganov BI, Samoilov AM, DI KleimenovSYu Levitsky, Yudin IK, Timofeeva AC, Muranov KO, Ostrovsky MA (2005) Mechanism of chaperone-like activity. Suppression of thermal aggregation of beta-l-crystallin by alpha-crystallin. Biochemistry 44:15480–15487

    Article  CAS  PubMed  Google Scholar 

  • Kublanov IV, Bidjieva SKh, Mardanov AV, Bonch-Osmolovskaya EA (2009) Desulfurococcus kamchatkensis sp. nov., a novel hyperthermophilic protein-degrading archaeon isolated from a Kamchatka hot spring. Int J Syst Evol Microbiol 59:1743–1747

    Article  CAS  PubMed  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Lobley A, Whitmore L, Wallace BA (2002) DICHROWEB: an interactive website for the analysis of protein secondary structure from circular dichroism spectra. Bioinformatics 18:211–212

    Article  CAS  PubMed  Google Scholar 

  • Markov DI, Zubov EO, Nikolaeva OP, Kurganov BI, Levitsky DI (2010) Thermal denaturation and aggregation of myosin subfragment 1isoforms with different essential light chains. Int J Mol Sci 11(11):4194–4226

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Markowitz VM, Chen IA, Palaniappan K, Chu K, Szeto E, Pillay M, Ratner A, Huang J, Woyke T, Hunteman M, Anderson I, Billis K, Varghese N, Mavromatis K, Pati A, Ivanova NN, Kyrpides NC (2014) IMG 4 version of the integrated microbial genomes comparative analysis system. Nucleic Acids Res 42:D560–D567 (Database issue)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Matsui M, Fowler JH, Walling LL (2006) Leucine aminopeptidases: diversity in structure and function. Biol Chem 387:1535–1544

    Article  CAS  PubMed  Google Scholar 

  • Permyakov EA (2003) The method of intrinsic protein luminescence. Nauka, Moscow

    Google Scholar 

  • Permyakov EA, Burstein EA (1984) Some aspects of studies of thermal transitions in proteins by means of their intrinsic fluorescence. Biophys Chem 19:265–271

    Article  CAS  PubMed  Google Scholar 

  • Petrova TE, Slutskaya ES, Boyko KM, Sokolova OS, Rakitina TV, Korzhenevskiy DA, Gorbacheva MA, Bezsudnova EY, Popov VO (2015) Structure of the dodecamer of the aminopeptidase APDkam598 from the archaeon Desulfurococcus kamchatkensis. Acta Cryst F 71:277–285

    Article  CAS  Google Scholar 

  • Privalov PL, Potekhin SA (1986) Scanning microcalorimetry in studying temperature-induced changes in proteins. Methods Enzymol 13:4–51

    Article  Google Scholar 

  • Provencher SW, Glockner J (1981) Estimation of globular protein secondary structure from circular dichroism. Biochemistry 20:33–37

    Article  CAS  PubMed  Google Scholar 

  • Ravin NV, Mardanov AV, Beletsky AV, Kublanov IV, Kolganova TV, Lebedinsky AV, Chernyh NA, Bonch-Osmolovskaya EA, Skryabin KG (2009) Complete genome sequence of the anaerobic, protein-degrading hyperthermophilic crenarchaeon Desulfurococcus kamchatkensis. J Bacteriol 191:2371–2379

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Rawlings ND, Morton FR (2008) The MEROPS batch BLAST: a tool to detect peptidases and their non-peptidase homologues in a genome. Biochimie 90:243–259

    Article  CAS  PubMed  Google Scholar 

  • Remaut H, Bompard-Gilles C, Goffin C, Frère JM, Van Beeumen J (2001) Structure of the Bacillus subtilis D-aminopeptidase DppA reveals a novel self-compartmentalizing protease. Nat Struct Biol 8:674–678

    Article  CAS  PubMed  Google Scholar 

  • Samanta U, Pal D, Chakrabarti P (2000) Environment of tryptophan side chains in proteins. Proteins 38:288–300

    Article  CAS  PubMed  Google Scholar 

  • Schoehn G, Vellieux FM, Dura MA, Receveur-Brechot V, Fabry CM, Ruigrok RW, Ebel C, Roussel A, Franzetti B (2006) An archaeal peptidase assembles into two different quaternary structures: a tetrahedron and a giant octahedron. J Biol Chem 281:36327–36337

    Article  CAS  PubMed  Google Scholar 

  • Slutskaya ES, Bezsudnova EY, Mardanov AV, Gumerov VM, Rakitina TV, Popov VO, Lipkin VM (2012) Characterization of a novel M42 aminopeptidase from crenarchaeon Desulfurococcus kamchatkensis. Dokl Biochem Biophys 442:30–32

    Article  CAS  PubMed  Google Scholar 

  • Sreerama N, Woody RW (2000) Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 287:252–260

    Article  CAS  PubMed  Google Scholar 

  • Staiano M, Scognamiglio V, Rossi M, D’Auria S, Stepanenko OV, Kuznetsova IM, Turoverov KK (2005) Unfolding and refolding of the glutamine-binding protein from Escherichia coli and its complex with glutamine induced by guanidine hydrochloride. Biochemistry 44:5625–5633

    Article  CAS  PubMed  Google Scholar 

  • Tamura T, Tamura N, Cejka Z, Hegerl R, Lottspeich F, Baumeister W (1996) Tricorn protease—the core of a modular proteolytic system. Science 274:1385–1389

    Article  CAS  PubMed  Google Scholar 

  • Taylor A (1993) Aminopeptidases: towards a mechanism of action. Trends Biochem Sci 18:167–171

    CAS  PubMed  Google Scholar 

  • Towell JF, Manning MC (1994) Analysis of protein structure by circular dichroism spectroscopy. In: Purdie N, Brittain HG (eds) Analytical applications of circular dichroism. Elsevier, Amsterdam, pp 175–207

    Chapter  Google Scholar 

  • Turoverov KK, Kuznetsova IM (2003) Intrinsic fluorescence of actin. J Fluoresc 13:41–57

    Article  CAS  Google Scholar 

  • Turoverov KK, Khaitlina SYu, Pinaev GP (1976) Ultra-violet fluorescence of actin. Determination of native actin content in actin preparations. FEBS Lett 62:4–6

    Article  CAS  PubMed  Google Scholar 

  • Vivian JT, Callis PR (2001) Mechanisms of tryptophan fluorescence shifts in proteins. Biophys J 80(5):2093–2109

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Whitmore L, Wallace BA (2004) DICHROWEB, an online server for protein secondary structure analyses from circular dichroism spectroscopic data. Nucleic Acids Res 32:W668–W673

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Woody RW (1995) Circular dichroism. Methods Enzymol 246:34–71

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We thank Dr. D. I. Levitsky for useful discussion and valuable comments. The work was supported by the Russian Scientific Foundation (project no. 14-24-00172).

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Authors and Affiliations

  1. A.N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Leninsky pr. 33, Moscow, 119071, Russian Federation

    Elvira Slutskaya, Natalia Artemova, Sergey Kleymenov & Vladimir Popov

  2. N.K. Koltsov Institute of Developmental Biology, Russian Academy of Sciences, Vavilova str. 26, Moscow, 119334, Russian Federation

    Sergey Kleymenov

  3. Institute of Mathematical Problems of Biology, RAS, Institutskaja str., 4, Pushchino, 142290, Russian Federation

    Tatiana Petrova

  4. Russian National Research Centre “Kurchatov Institute”, Akademika Kurchatova sq. 1, Moscow, 123182, Russian Federation

    Vladimir Popov

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  1. Elvira Slutskaya
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  2. Natalia Artemova
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  4. Tatiana Petrova
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  5. Vladimir Popov
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Correspondence to Elvira Slutskaya.

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Slutskaya, E., Artemova, N., Kleymenov, S. et al. Heat-induced conformational changes of TET peptidase from crenarchaeon Desulfurococcus kamchatkensis . Eur Biophys J 44, 667–675 (2015). https://doi.org/10.1007/s00249-015-1064-3

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  • DOI: https://doi.org/10.1007/s00249-015-1064-3

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