The Protein Journal

, Volume 31, Issue 1, pp 93–107 | Cite as

Expression, Purification, Structural and Functional Analysis of SycB: A Type Three Secretion Chaperone From Yersinia enterocolitica

Article
First Online:

Abstract

In Yersinia enterocolitica biovar 1B, a genome encoded TTSS designated as Ysa-Ysp system is used for virulence. SycB is an annotated chaperone to this system. SycB is soluble in presence of translocator YspC. SycB and its truncated form (∆SycB(1–114)) exist as dimers. YspC forms a 1:1 complex with SycB. Homology model of SycB shows a flexible N-terminal may be required for solubility and dimerization; and concave core formed by antiparallel helices of TPRs. Far UV CD spectra confirm that SycB is predominantly alpha helical. Near UV CD spectra show that SycB has tertiary structure at pH 7.2 (native folded protein), which disappears at pH 5 (molten globule) and SycB releases YspC at pH 5. SycB has a cooperative melting behavior. At pH 7.2, SycB shows solvent accessible hydrophobic patches. Concave core in the model exhibits ANS binding within FRET distance of tyrosines in the TPR, allowing a range of interaction of SycB with its ligand.

Keywords

Class II chaperone Homology model TPR helices pH and protein folding Molten Globule Intrinsic and extrinsic fluorophore ANS quenching and FRET 

Abbreviations

TTSS

Type three secretion system

Ysa-Ysp

Yersinia secretion apparatus-Yersinia secretion protein

TPR

Tetratricopeptide repeat

CD

Circular dichroism

ANS

8-anilinonapthalene-1-sulfonate

FRET

Forsters resonance energy transfer

Ysc-Yop

Yersinia secretion component-Yersinia outer protein

DLS

Dynamic light scattering

BLAST

Basic Local Search Alignment Tool

NCBI

National Center for Biotechnology Information

MSA

Multiple sequence alignment

PDB

Protein Data Bank

ATCC

American Type Culture Collection

MCS

Multiple cloning site

LB

Luria Bertani

IPTG

Isopropyl β-D-1-thiogalactopyranoside

PMSF

Phenylmethanesulfonyl fluoride

Ni–NTA

Nickel–nitrilotriacetic acid

CHCA

α-cyano hydroxyl cinnamic acid

MALDI

Matrix assisted laser desorption ionization

EDTA

Ethylenediaminetetraacetic acid

EGS

Ethylene Glycol bis[succinimidyl succinate]

TOF

Time of flight

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Notes

Acknowledgments

The research was funded by grants from the Department of Science and Technology, Government of India. Indian Institute of Chemical Biology (IICB), a unit of Council of Scientific and Industrial Research provided the facilities and fellowships for this research.

Supplementary material

10930_2011_9377_MOESM1_ESM.doc (166 kb)
Supplementary material 1 (DOC 166 kb)

References

  1. 1.
    Barta ML, Zhang L, Picking WL, Geisbrecht BV (2010) BMC Struct Biol 10:21CrossRefGoogle Scholar
  2. 2.
    Bekard IB, Dunstan DE (2009) Biophys J 97(9):2521–2531CrossRefGoogle Scholar
  3. 3.
    Birket SE, Harrington AT, Espina M, Smith ND, Terry CM, Darboe N, Markham AP, Middaugh CR, Picking WL, Picking WD (2007) Biochemistry 46(27):8128–8137CrossRefGoogle Scholar
  4. 4.
    Büttner CR, Sorg I, Cornelis GR, Heinz DW, Niemann HH (2008) J Mol Biol 375(4):997–1012CrossRefGoogle Scholar
  5. 5.
    Carter PB (1975) Infect Immun 11(1):164–170Google Scholar
  6. 6.
    Chen RF, Cohen PF (1966) Arch Biochem Biophys 114(3):514–522CrossRefGoogle Scholar
  7. 7.
    Clark MA, Hirst BH, Jepson MA (1998) Infect Immun 66:1237–1243Google Scholar
  8. 8.
  9. 9.
  10. 10.
    Conserved Domain Database [http://www.ncbi.nlm.nih.gov/cdd/]
  11. 11.
    Cornelis GR, Boland A, Boyd AP, Geuijen C, Iriarte M, Neyt C, Sory MP, Stainier I (1998) Microbiol Mol Biol Rev 62:1315–1352Google Scholar
  12. 12.
    Cornelis GR, Laroche Y, Balligand G, Sory MP, Wauters G (1987) Rev Infect Dis 9:64–87CrossRefGoogle Scholar
  13. 13.
    Cornelis GR, Wolf-Watz H (1997) Mol Microbiol 23:861–867CrossRefGoogle Scholar
  14. 14.
    Corpet F (1988) Nucleic Acids Res 16(22):10881–10890CrossRefGoogle Scholar
  15. 15.
    Cover TL, Aber RC (1989) N Engl J Med 321:16–24CrossRefGoogle Scholar
  16. 16.
    Crooks GE, Hon G, Chandonia JM, Brenner SE (2004) Genome Res 14:1188–1190CrossRefGoogle Scholar
  17. 17.
    D’Andrea LD, Regan L (2003) Trends Biochem Sci 28:655–662CrossRefGoogle Scholar
  18. 18.
    Das AK, Cohen PW, Barford D (1998) EMBO J 17:1192–1199CrossRefGoogle Scholar
  19. 19.
    Edqvist PJ, Broms JE, Betts HJ, Forsberg A, Pallen MJ, Francis MS (2006) Mol Microbiol 59(1):31–44CrossRefGoogle Scholar
  20. 20.
    Faudry E, Job V, Dessen A, Attree I, Forge V (2007) FEBS J 274(14):3601–3610CrossRefGoogle Scholar
  21. 21.
    Foultier B, Troisfontaines P, Muller S, Opperdoes FR, Cornelis GR (2002) J Mol Evol 55:37–51CrossRefGoogle Scholar
  22. 22.
    Foultier B, Troisfontaines P, Vertommen D, Marenne MN, Rider M, Parsot C, Cornelis GR (2003) Infect Immun 71(1):242–253CrossRefGoogle Scholar
  23. 23.
    Gouet P, Courcelle E, Stuart D, Metoz F (1999) Bioinformatics 15(4):305–308CrossRefGoogle Scholar
  24. 24.
    Grutzkau A, Hanski C, Hahn H, Riecken E (1990) Gut 31:1011–1015CrossRefGoogle Scholar
  25. 25.
    Guzow K, Szabelski M, Rzeska A, Karolczak J, Sulowska H, Wiczk W (2002) Chem Phys Lett 362:519–526CrossRefGoogle Scholar
  26. 26.
    Håkansson S, Bergman T, Vanooteghem JC, Cornelis G, Wolf-Watz H (1993) Infect Immun 61:71–80Google Scholar
  27. 27.
    Haller JC, Carlson S, Pederson KJ, Pierson DE (2000) Mol Microbiol 36:1436–1446CrossRefGoogle Scholar
  28. 28.
    Hamada D, Kato T, Ikegami T, Suzuki KN, Hayashi M, Murooka Y, Honda T, Yanagihara I (2005) FEBS J 272(3):756–768CrossRefGoogle Scholar
  29. 29.
    Hanski C, Kutschka U, Schmoranzer HP, Naumann M, Stallmach A, Hahn H, Menge H, Riecken EO (1989) Infect Immun 57:673–678Google Scholar
  30. 30.
    Job V, Matteï PJ, Lemaire D, Attree I, Dessen A (2010) J Biol Chem 285(30):23224–23232CrossRefGoogle Scholar
  31. 31.
  32. 32.
    Kato T, Hamada D, Fukui T, Hayashi M, Honda T, Murooka Y, Yanagihara I (2005) FEBS J 272(11):2773–2783CrossRefGoogle Scholar
  33. 33.
    Lunelli M, Lokareddy RK, Zychlinsky A, Kolbe M (2009) Proc Natl Acad Sci 106:9661–9666CrossRefGoogle Scholar
  34. 34.
    Matsumoto H, Young GM (2006) Mol Microbiol 59(2):689–706CrossRefGoogle Scholar
  35. 35.
    Ménard R, Sansonetti PJ, Parsot C, Vasselon T (1994) Cell 79:515–525CrossRefGoogle Scholar
  36. 36.
    Mildiner-Earley S, Walker KA, Miller VL (2007) Adv Exp Med Biol 603:211–216CrossRefGoogle Scholar
  37. 37.
    Murali J, Jayakumar R (2005) J struct Biol 150:180–189CrossRefGoogle Scholar
  38. 38.
  39. 39.
    Pallen MJ, Francis MS, Fütterer K (2003) FEMS Microbiol Lett 223(1):53–60CrossRefGoogle Scholar
  40. 40.
    Parsot C, Hamiaux C, Page AL (2003) Curr Opin Microbiol 6:7–14CrossRefGoogle Scholar
  41. 41.
    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) J Comput Chem 25(13):1605–1612CrossRefGoogle Scholar
  42. 42.
    Picking WL, Mertz JA, Marquart ME, Picking WD (1996) Protein Expr Purif 8(4):401–408CrossRefGoogle Scholar
  43. 43.
  44. 44.
    Ptitsyn OB (1995) Advs Protein Chem 47:83–229CrossRefGoogle Scholar
  45. 45.
    Roy A, Kucural A, Zhang Y (2010) Nat Protoc 5(4):725–738CrossRefGoogle Scholar
  46. 46.
    Schoehn G, Guilmi AMD, Lemaire D, Attree I, Weissenhorn W, Dessen A (2003) EMBO J 22(19):4957–4967CrossRefGoogle Scholar
  47. 47.
    Tan YW, Yu HB, Sivaraman J, Leung KY, Mok YK (2009) Protein Sci 18:1724–1734CrossRefGoogle Scholar
  48. 48.
    The Chimera home page http://www.cgl.ucsf.edu/chimera
  49. 49.
    The PyMOL Molecular graphics system [http://www.PyMOL.org]
  50. 50.
  51. 51.
    Venecia K, Young GM (2005) Infect Immun 73(9):5961–5977CrossRefGoogle Scholar
  52. 52.
    Walker KA, Miller VL (2004) J Bacteriol 186(13):4056–4066CrossRefGoogle Scholar
  53. 53.
    Walker KA, Miller VL (2009) J Bacteriol 191(6):1816–1826CrossRefGoogle Scholar
  54. 54.
    Walker KA, Obrist MW, Shirly Mildiner-Earley S, Miller VL (2010) J Bacteriol 192(22):5887–5897CrossRefGoogle Scholar
  55. 55.
    Whitmore L, Wallace BA (2004) Nucleic Acid Res 32:668–673CrossRefGoogle Scholar
  56. 56.
    Young GM (2007) Adv Exp Med Biol 603:286–297CrossRefGoogle Scholar
  57. 57.
    Yu XJ, McGourty K, Liu M, Unsworth KE, Holden DW (2010) Science 328:1040–1043CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Abhishek Basu
    • 1
  • Rakesh Chatterjee
    • 1
  • Saumen Datta
    • 1
  1. 1.Structural Biology and Bioinformatics DivisionIndian Institute of Chemical BiologyKolkataIndia

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