Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Application of Temperature Gradient Gel Electrophoresis To cAMP Receptor Protein

온도 기울기 전기영동장치의 CAMP 수용성 단백질에 응용

  • Published : 2004.04.01
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Abstract

Cyclic AMP receptor protein (CRP) is involved in the transcriptional regulation of more than 100 genes in E. coli. CRP dimer is converted into active form via the sequential conformation change of cAMP binding pocket, hinge region and HTH DNA binding motif by binding of cAMP. The temperature gradient gel electrophoresis (TGGE) was applied to CRP protein to know whether it was an efficient technique to study the conformational transitions and the thermal stability. TGGE showed the unfolding process of wild-type and S83G CRP proteins with the temperature gradient set from 29 to 71$^{\circ}C$ on nondenaturing polyacrylamide gel. Melting temperature (Tm) was 57$\pm$1 and 55$\pm$1$^{\circ}C$ for wild-type and S83G CRP, respectively in acidic buffer[89.8 mM Glycine and 24 mM Boric acid (pH 5.8)].

cAMP수용성 단백질(CRP)은 E. coli의 100가지 이상의 유전자 전자조절에 관계된다. CRP는 dimer로 존재하며 cAMP의 결합으로 활성인 형태로 전환된다. 이중체인 CRP 단백질의 열 안정성과 구조 전이의 연구에 효과적인 온도 기울기 전기영동장치를 이용하여 확인하였다. 본 연구에서 야생형과 S83C CRP 단백질의 melting temperature (Tm)는 산성인 완충용액[89.8 mM Glycine, 24mM Boric acid (pH 5.8)]에서 57$\pm$1(야생형 CRP)과 55$\pm$1$^{\circ}C$ (S83G CRP)였다. 그리고 온도에 따른 CRP 단백질의 구조변화도 protease digestion과 CD spectropolarimeter을 이용하여 확인하였다.

Keywords

References

  1. Nucleic Acid Res. v.10 Molecular cloning and nucleotide sequencing of the gene for E. coli cAMP receptor protein Aiba,H.;S.Fujimoto;N.Ozaki https://doi.org/10.1093/nar/10.4.1345
  2. Anal. Biochem. v.208 Analysis of the heat-induced denaturation of proteins using temperature gradient gel electrophoresis Arakawa,T.;L.Hung;V.Pan;T.P.Horan;C.G.Kolvenbach;L.O.Narhi https://doi.org/10.1006/abio.1993.1042
  3. Biochemistry v.40 Amino acid substitution at position 99 affetcts the rate of CRP subunit exchange Baker,C.H.;S.R.Tomlinson;A.E.Garcia;J.G.Harman https://doi.org/10.1021/bi010834+
  4. Electrophoresis v.11 Analysis of the conformational transitions of proteins by temperature-gradient gel electrophoresis Birmes,A.;A.Sattles;K.H.Maurer;D.Riesner https://doi.org/10.1002/elps.1150111004
  5. Nature v.326 Knowledge-based prediction of protein structures and the design of novel molecules Blundell,T.L.;B.L.Sibanda;M.J.Sternberg;J.M.Thornton https://doi.org/10.1038/326347a0
  6. Microbiological Reviews v.56 Cyclic AMP in prokaryotes Botsford,J.L.;J.G.Harman
  7. Nucleic Acids Res. v.10 Cloning and sequence of the crp gene of Escherichia coli K 12 Cossart,P.;B.Gicquel-Sanzey https://doi.org/10.1093/nar/10.4.1363
  8. J. Biol. Chem. v.261 Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein Harman,J.G.;K.McKenney;A.Peterkofsky
  9. Korean Journal of Life Science v.10 Study on the sturcture of cAMP receptor protein (CRP) by temperature change Joo,J.;M.J.Goo;J.Gang
  10. Electrophoresis v.16 Analysis of thyroid stimulating hormone-receptor mutations by temperature-gradient gel electrophoresis Koch,M.;R.Wahl;F.J.Seif https://doi.org/10.1002/elps.11501601121
  11. Nucleic Acids Res. v.22 Mutagenesis of tye cyclic AMP receptor protein of Escherichia coli: targeting positions 83, 127 and 128 of the cyclic nucleotide binding pocket Lee,E.J.;J.Glasgow;S.F.Leu;A.O.Belduz;J.G.Harman https://doi.org/10.1093/nar/22.15.2894
  12. Eur. J. Biochem. v.243 Stability and kinetics of unfolding and refolding of cAMP receptor protein from Escherichia coli Malecki,J.;Z.Wasylewski https://doi.org/10.1111/j.1432-1033.1997.00660.x
  13. Biophys. Chem. v.26 Temperature-gradient gel electrophoresis. Thermoynamic analysis of nucleic acids and proteins in purified form and in cellular extracts Rosenbaum,V.;D.Riesner https://doi.org/10.1016/0301-4622(87)80026-1
  14. Electrophoresis v.14 Temperature-gradient gel electrophoresis for analysis and screening of thermostable proteases Sattler,A.;D.Riesner https://doi.org/10.1002/elps.11501401122
  15. Biochemistry v.39 Intersubunit association induces unique allosteric dependence of the T127L CRP mutant on pH Shi,Y.;S.Wang;F.P.Schwarz https://doi.org/10.1021/bi000225m
  16. Electrophoresis v.21 Heat-induced conformational transition of cytochrome c observed by temperature gradient gel electrophoresis at acidic pH Viglasky,V.;M.Antalik;J.Bagel'ova;Z.Tomori;D.Podhrasky https://doi.org/10.1002/(SICI)1522-2683(20000301)21:5<850::AID-ELPS850>3.0.CO;2-4