미생물학회지 (Korean Journal of Microbiology)

  • 제53권4호
  • /
  • Pages.297-303
  • /
  • 2017
  • /
  • 0440-2413(pISSN)
  • /
  • 2383-9902(eISSN)
한국미생물학회 (The Microbiological Society of Korea)
권호 표지
DOI QR코드

DOI QR Code

교차결합 실험을 통한 루신 대응 조절 단백질의 4차 구조 분석

Analysis of quaternary structure of leucine-responsive regulatory protein (Lrp) by crosslink experiments

  • Lee, Euiho (Department of Biochemistry, Chungnam National University) ;
  • Pokoo, Robert (Department of Biochemistry, Chungnam National University) ;
  • Nguyen, Loi Thuan (Department of Biochemistry, Chungnam National University) ;
  • Lee, Chan Yong (Department of Biochemistry, Chungnam National University)
  • 투고 : 2017.08.23
  • 심사 : 2017.11.22
  • 발행 : 2017.12.31
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

루신-대응 조절 단백질(Lrp)은 18.8 kDa의 분자량을 갖는 글로벌 조절 단백질로서 대장균과 같은 장내세균과에서 많은 대사작용 오페론의 기능적 활성도를 조절한다. 단백질의 4차 구조를 규명하기 위한 목적으로 Lrp단백질 코드하는 유전자가 삽입된 재조합 플라스미드 pQE vector를 발현시킨 6 ${\times}$ His-tag Lrp 야생형과 $^3H$로 표지된 Lrp를 분리 정제한 후 cross linker들인 glutaraldehyde, 1,2,3,4-diepoxy-butane (DEB), ethylene glycol bis (succinimidyl succinate) (EGS)으로 cross link 실험을 수행하여 Lrp가 $0.3{\mu}M$ 이하의 낮은 농도에서나 $5{\mu}M$의 높은 농도에서 이량체, 사량체, 육량체, 팔량체로 존재할 수 있음을 확인하였다.

Leucine-responsive regulatory protein (LRP) is a regulatory protein of molecular weight 18.8 kDa and is widely known to regulate many metabolic and functional activities of operons in Escherichia coli. The gene for Lrp from Escherichia coli in pQE system of 6 ${\times}$ His-tagging was expressed and $^3H$-labeled protein, as well as the wild type Lrp, was purified. The crosslink experiments were performed to analyze the quaternary structure of Lrp at high of $5{\mu}M$ and at low concentrations below $0.3{\mu}M$ with cross linkers, such as glutaraldehyde, 1, 2, 3, 4-diepoxy-butane (DEB), and ethylene glycol bis (succinimidyl succinate) (EGS). In the experiments, we found that the Lrp protein can be formed higher conformation states of tetramer, hexamer, octamer, as well as dimeric state when incubated with the above cross linkers.

키워드

참고문헌

  1. Barton, G.J. 1993. ALSCRIPT: a tool to format multiple sequence alignments. Protein Eng. 6, 37-40. https://doi.org/10.1093/protein/6.1.37
  2. Brinkman, A.B., Ettema, G.J.T., de Vos, W.M., and Van der Oost, J. 2003. The Lrp family of transcriptional regulators. Mol. Microbiol. 48, 287-294. https://doi.org/10.1046/j.1365-2958.2003.03442.x
  3. Calvo, J.M. and Matthews, R.G. 1994. The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli. Microbiol. Rev. 58, 466-490.
  4. Chen, S. and Calvo, J.M. 2002. Leucine-induced dissociation of Escherichia coli Lrp hexadecamers to octamers. J. Mol. Biol. 318, 1031-1042. https://doi.org/10.1016/S0022-2836(02)00187-0
  5. Chen, S., Rosner, M.H., and Calvo, J.M. 2001. Leucine-regulated self-association of leucine-responsive regulatory protein (Lrp) from Escherichia coli. J. Mol. Biol. 312, 625-635. https://doi.org/10.1006/jmbi.2001.4955
  6. Chen, S., Iannolo, M., and Calvo, J.M. 2005. Cooperative binding of the leucine-responsive regulatory protein (Lrp) to DNA. J. Mol. Biol. 345, 251-264. https://doi.org/10.1016/j.jmb.2004.10.047
  7. Cui, Y., Midkiff, M.A., Wang, Q., and Calvo, J.M. 1996. The leucine responsive regulatory protein (Lrp) from Escherichia coli. Stoichiometry and minimal requirements for binding to DNA. J. Biol. Chem. 271, 6611-6617. https://doi.org/10.1074/jbc.271.12.6611
  8. Cho, B.K., Barett, C.L., Knight, E.M., Park, Y.S., and Palasson, B.O. 2008. Genome scale reconstruction of the Lrp regulatory network in Escherichia coli. Proc. Natl. Acad. Sci. USA 105, 19462-19467. https://doi.org/10.1073/pnas.0807227105
  9. Deng, W., Wang, H., and Xie, J. 2011. Regulatory and pathogenesis roles of Mycobacterium Lrp/AsnC family transcriptional factors. J. Cell Biochem. 112, 2655-2662. https://doi.org/10.1002/jcb.23193
  10. de los Rios, S. and Perona, J.J. 2007. Structure of the Escherichia coli leucine-responsive regulatory protein (Lrp) reveals a novel octameric assembly. J. Mol. Biol. 366, 1589-1602. https://doi.org/10.1016/j.jmb.2006.12.032
  11. Ernsting, B.R., Atkinson, M.R., Matthews, R.G., and Ninfa, A.J. 1992. Characterization of the regulon controlled by the leucine responsive regulatory protein in Escherichia coli. J. Bacteriol. 174, 1109-1118. https://doi.org/10.1128/jb.174.4.1109-1118.1992
  12. Ettema, T.J.G., Brinkman, A.B., Tani, T.H., Rafferty, J.B., and van der Oost, J. 2002. A novel ligand-binding domain involved in regulation of amino acid metabolism in prokaryotes. J. Biol. Chem. 277, 37464-37468. https://doi.org/10.1074/jbc.M206063200
  13. Hart, B.R. and Blumenthal, R.M. 2011. Unexpected coregulatory range for the global regulator Lrp of Escherichia coli and Proteus mirabillis. J. Bacteriol. 193, 1054-1064. https://doi.org/10.1128/JB.01183-10
  14. Hart, B.R., Mishra, P.K., Listner, R.E., Hinerman, J.M., Herr, A.B., and Blumenthal, R.M. 2011. Recognition of DNA by the helix-turn-helix global regulatory protein Lrp is modulated by the amino-terminus. J. Bacteriol. 193, 3794-3803. https://doi.org/10.1128/JB.00191-11
  15. Hung, S., Pierre, B., and Wesley, H.G. 2002. Global gene expression profiling in Escherichia coli K-12. The effects of leucineresponsive regulatory protein. J. Biol. Chem. 277, 40309-40323. https://doi.org/10.1074/jbc.M204044200
  16. Landgraf, J.R., Wu, J., and Calvo, J.M. 1996. Effects of nutrition and growth rate on Lrp levels in Escherichia coli. J. Bacteriol. 178, 6930-6936. https://doi.org/10.1128/jb.178.23.6930-6936.1996
  17. Lee, C.Y., Kim, S.C., and Seo, C.H. 2010. Purification and fluorometric analysis of leucine-responsive regulatory protein from Escherichia coli. Korean J. Microbiol. 46, 104-108.
  18. Newman, E.B., D'Ari, R., and Lin, R.T. 1992. The leucine-Lrp regulon in Escherichia coli: A global response in search of a raison d'etre. Cell 68, 618-620.
  19. Newman, E.B, Lin, R.T., and D'Ari, R. 1996. The leucine/Lrp regulon. Annu. Rev. Microbiol. 49, 1513-1525.
  20. Parry-Smith, D.J., Payne, A.W.R., Mitchie, A.D., and Attwood, T.K. 1998. CINEMA-a novel colour interactive editor for multiple alignments. Gene 221, 57-63.
  21. Peterson, S.N., Dahlquist, F.W., and Reich, N.O. 2007. The role of high affinity non-specific DNA binding by Lrp in transcriptional regulation and DNA organization. J. Mol. Biol. 369, 1307-1317. https://doi.org/10.1016/j.jmb.2007.04.023
  22. Philip, M.L., Sander, H.J.S., Svetlana, E.S., Arie, B.B., Willem, M.D., Van der Oost, J., David, W.R., and John, B.R. 2001. Crystal structure of the Lrp-like transcriptional regulator from the archaeon Pyrococcus furiosus. EMBO J. 20, 990-997. https://doi.org/10.1093/emboj/20.5.990
  23. Pokoo, R., Lee, E.H., and Lee, C.Y. 2014. Fluorescence characteristics of a tryptophan mutant of leucine-responsive regulatory protein. Korean J. Microbiol. 50, 275-280. https://doi.org/10.7845/kjm.2014.4075
  24. Shimada, T., Saito, N., Maeda, M., Tanaka, K., and Ishihama, A. 2015. Expanded roles of leucine responsive regulatory protein in transcription regulation of the Escherichia coli gemome: Genomic SELEX screening regulation targets. Microb. Genom. 1, e000001.
  25. Wang, Q. and Calvo, J.M. 1993. Lrp, a global regulatory protein of Escherichia coli, binds cooperatively to multiple sites and activates transcription of ilvIH. J. Mol. Biol. 229, 306-318. https://doi.org/10.1006/jmbi.1993.1036