Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지
DOI QR코드

DOI QR Code

Fluorescence Characteristics of a Tryptophan Mutant of Leucine-responsive Regulatory Protein (Lrp)

트립토판 돌연변이 루신-반응 조절 단백질의 형광 특성

  • Pokoo, Robert (Department of Biochemistry, Chungnam National University) ;
  • Lee, Eui Ho (Department of Biochemistry, Chungnam National University) ;
  • Lee, Chan Yong (Department of Biochemistry, Chungnam National University)
  • Received : 2014.11.10
  • Accepted : 2014.12.03
  • Published : 2014.12.31
Download PDF
⟨ Previous Next ⟩

Abstract

Leucine-responsive Regulatory Protein (Lrp) from Escherichia coli is an 18.8 kDa protein composed of 164 amino acids. Wild type Lrp (Lrp Wt) does not possess any tryptophan amino acid which has strong intrinsic fluorescence, whereas the mutant Lrp R145W contains a single tryptophan at the position 145 in the leucine-responsive domain. To investigate the fluorescence character, the Lrp R145W and Lrp Wt proteins were purified. The fluorescence intensity of Lrp R145W is much higher than that of wild type protein, and the intensity of Lrp R145W was decreased by binding to its specific DNA designed from ilvIH operon and to L-leucine. In addition, the tryptophan fluorescence intensity of Lrp R145W was strongly quenched by addition of acrylamide even in the least amount of concentration as well as by urea. The data obtained from this study may give valuable information on the three dimensional structure of Lrp R145W.

루신-반응 조절 단백질(Lrp)은 18.8 kDa의 분자량을 갖는 164개의 아미노산으로 이루어진 글로벌 조절 단백질으로서, 야생형의 단백질(Lrp Wt)에는 아미노산 중 가장 강한 자체 형광을 띠는 트립토판이 존재하지 않는다. Lrp 단백질의 구조변이에 대한 정보를 줄 수 있는 형광분석을 위하여 Lrp Wt과 트립토판이루신-반응 영역에 단지 하나 존재하는 돌연변이 단백질(Lrp R145W)을 분리 정제하였다. Lrp R145W 단백질은 이들 ilvIH 오페론에서 고안된 Lrp 결합 특정 DNA와 아미노산 루신과의 결합 후에 형광이 감소하였으며 acrylamide, urea 등에 의해서도 급격히 쇄광하는 양상을 보였다. 이들 형광 실험 결과는 Lrp의 3차원적 구조 및 배향을 연구에 중요한 정보를 제공하여 줄 수 있을 것이다.

Keywords

References

  1. 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
  2. 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.
  3. 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
  4. 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
  5. 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
  6. 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
  7. 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
  8. Lackowicz, J.R. 1983. Principles of fluorescence spectroscopy. pp. 111- 153. Plenum press, New York, USA.
  9. 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
  10. Lee, C.Y., Kim, S.C., and Seo, C.H. 2010. Purification and fluorometric analysis of leucine-responsive regulatory protein from Escherichia coli. Kor. J. Microbiol. 46, 104-108.
  11. 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.
  12. Newman, E.B. and Lin, R. 1995. Leucine-responsive regulatory protein: a global regulator of gene expression in Escherichia coli. Annu. Rev. Microbiol. 49, 747-775. https://doi.org/10.1146/annurev.mi.49.100195.003531
  13. Newman, E.B., Lin, R.T., and D.Ari, R. 1996. The leucine/Lrp regulon. Annu. Rev. Microbiol. 49, 1513-1525.
  14. 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
  15. Rica, E., Aker, D.A., and Calvo, J.M. 1989. A protein that binds to the regulatory region of the ilvIH operon of Escherichia coli. J. Bacteriol. 171, 1658-1664. https://doi.org/10.1128/jb.171.3.1658-1664.1989
  16. Roesch, P.L. and Bloomfield, I.C. 1998. Leucine alters the interaction of the leucine-responsive regulatory protein (Lrp) with the firm switch to stimulate site-specific recombination in Escherichia coli. Mol. Microbiol. 27, 751-761. https://doi.org/10.1046/j.1365-2958.1998.00720.x
  17. She-pin, H., Pierre, B., and Wesley, H.G. 2002. Global gene expression profiling in Escherichia coli K-12. The effects of Leucine-responsive regulatory protein. J. Biol. Chem. 277, 40309-40323. https://doi.org/10.1074/jbc.M204044200
  18. Thaw, P., Sedelnikova, S.E., Muranova, T., Wiese, S., Ayora, S., Alonso, J.C., Brinkman, A.B, Akerboom, J., van der Oost, J., and Rafferty, J.B. 2003. Structural insight into gene transcriptional regulation and effector binding by the Lrp/AsnC family. Nucleic Acids Res. 34, 1439-1449.
  19. 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
  20. Willins, D.A., Ryan, C.W., Platko, J.V., and Calvo, J.M. 1991. Characterization of Lrp, an Escherichia coli regulatory protein that mediates a global response to leucine. J. Biol. Chem. 26, 10768- 10774.

Cited by

  1. Analysis of quaternary structure of leucine-responsive regulatory protein (Lrp) by crosslink experiments vol.53, pp.4, 2014, https://doi.org/10.7845/kjm.2017.7063