DOI QR코드

DOI QR Code

Folding Mechanism of WT* Ubiquitin Variant Studied by Stopped-flow Fluorescence Spectroscopy

  • Park, Soon-Ho (Department of Biochemistry and Molecular Biology, Research Institute of Oral Sciences, College of Dentistry, Gangneung-Wonju National University)
  • Received : 2010.07.21
  • Accepted : 2010.08.23
  • Published : 2010.10.20

Abstract

The folding kinetics of $WT^*$ ubiquitin variant with valine to alanine mutation at sequence position 26 (HubWA) was studied by stopped-flow fluorescence spectroscopy. While unfolding kinetics showed a single exponential phase, refolding reaction showed three exponential phases. The semi-logarithmic plot of urea concentration vs. rate constant for the first phase showed v-shape pattern while the second phase showed v-shape with roll-over effect at low urea concentration. The rate constant and the amplitude of the third phase were constant throughout the urea concentrations, suggesting that this phase represents parallel process due to the configurational isomerization. Interestingly, the first and second phases appeared to be coupled since the amplitude of the second phase increased at the expense of the amplitude of the first phase in increasing urea concentrations. This observation together with the roll-over effect in the second folding phase indicates the presence of intermediate state during the folding reaction of HubWA. Quantitative analysis of Hub-WA folding kinetics indicated that this intermediate state is on the folding pathway. Folding kinetics measurement of a mutant HubWA with hydrophobic core residue mutation, Val to Ala at residue position 17, suggested that the intermediate state has significant amount of native interactions, supporting the interpretation that the intermediate is on the folding pathway. It is considered that HubWA is a useful model protein to study the contribution of residues to protein folding process using folding kinetics measurements in conjunction with protein engineering.

Keywords

References

  1. Matouschek, A.; Kellis, J. T., Jr.; Serrano, L.; Fersht, A. R. Nature 1989, 340, 122. https://doi.org/10.1038/340122a0
  2. Guydosh, N. R.; Fersht, A. R. In Protein Folding Handbook, Part 1; Keifhaber, T., Buchner, J., Eds.; Wiley-VCH: Weinhelm, Germany, 2005; p 445.
  3. Zarrine-Afsar, A.; Davidson, A. R. Methods 2004, 34, 41. https://doi.org/10.1016/j.ymeth.2004.03.013
  4. Fersht, A. R. Curr. Opin. Struct. Biol. 1995, 5, 79. https://doi.org/10.1016/0959-440X(95)80012-P
  5. Matouschek, A.; Kellis, J. T. J.; Serrano, L.; Bycroft, M.; Fersht, A. R. Nature 1990, 346, 440. https://doi.org/10.1038/346440a0
  6. Itzhaki, L. S.; Otzen, D. E.; Fersht, A. R. J. Mol. Biol. 1995, 254, 260. https://doi.org/10.1006/jmbi.1995.0616
  7. Lindorff-Larsen, K.; Vendruscolo, M.; Paci, E.; Dobson, C. M. Nat. Struct. & Mol. Biol. 2004, 11, 443. https://doi.org/10.1038/nsmb765
  8. Martinez, J. C.; Serrano, L. Nat. Struct. Biol. 1999, 6, (1010-1016). https://doi.org/10.1038/14896
  9. Northey, J. G. B.; Di Nardo, A. A.; Davidson, A. R. Nat. Struct. Biol. 2002, 9, 126. https://doi.org/10.1038/nsb748
  10. Riddle, D. S.; Grantcharova, V. P.; Santiago, J. V.; Alm, E.; Ruczincski, I.; Baker, D. Nat. Struct. Biol. 1999, 6, 1016. https://doi.org/10.1038/14901
  11. McCallister, E. L.; Alm, E.; Baker, D. Nat. Struct. Biol. 2000, 7, 669. https://doi.org/10.1038/77971
  12. Kim, D. E.; Fisher, C.; Baker, D. J. Mol. Biol. 2000, 298, 971. https://doi.org/10.1006/jmbi.2000.3701
  13. Kragelund, B. B.; Osmark, P.; Neergaard, T. B.; Schiodt, J.; Kristiansen, K.; Knudsen, J.; Poulsen, F. M. Nat. Struct. Biol. 1999, 6, 594. https://doi.org/10.1038/9384
  14. Khorasanizadeh, S.; Peters, I. D.; Butt, T. R.; Roder, H. Biochemistry 1993, 32, 7054. https://doi.org/10.1021/bi00078a034
  15. Jackson, S. E. Org. Biomol. Chem. 2006, 4, 1845. https://doi.org/10.1039/b600829c
  16. Khorasanizadeh, S.; Peters, I. D.; Roder, H. Nat. Struct. Biol. 1996, 3(2), 193. https://doi.org/10.1038/nsb0296-193
  17. Krantz, B. A.; Sosnick, T. R. Biochemistry 2000, 39, 11696. https://doi.org/10.1021/bi000792+
  18. Went, H. M.; Benitez-Cordoza, C. G.; Jackson, S. E. FEBS Lett. 2004, 567, 333. https://doi.org/10.1016/j.febslet.2004.04.089
  19. Silow, M.; Oliveberg, M. Proc. Natl. Acad. Sci. USA 1997, 94, 6084. https://doi.org/10.1073/pnas.94.12.6084
  20. Park, S.-H. BMB Reports 2008, 41, 35. https://doi.org/10.5483/BMBRep.2008.41.1.035
  21. Peterman, B. F. Anal. Biochem. 1979, 93, 442. https://doi.org/10.1016/S0003-2697(79)80176-1
  22. Bachmann, A.; Kiefhaber, T. In Protein Folding Handbook, Part I; Buchner, J., Kiefhaber, T., Eds.; WILEY-VCH: Weinheim, Germany, 2005; p 379.
  23. Tanford, C. Adv. Prot. Chem. 1970, 24, 1. https://doi.org/10.1016/S0065-3233(08)60241-7
  24. Chen, B.; Baase, W. A.; Schellman, J. A. Biochemistry 1989, 28, 691. https://doi.org/10.1021/bi00428a042
  25. Matthews, C. R. Methods Enzymol. 1987, 154, 498. https://doi.org/10.1016/0076-6879(87)54092-7
  26. Maxwell, K. L.; Wildes, D.; Zarrine-Afsar, A.; De Los Rios, M. A.; Brown, A. G.; Friel, C. T.; Hedberg, L.; Horng, J.-C.; Bona, D.; Miller, E. J.; Vallee-Belisle, A.; Main, E. R. G.; Bemporad, F.; Qiu, L.; Teilum, K.; Vu, N.-D.; Edwards, A. M.; Ruczinski, I.; Poulsen, F. M.; Kragelund, B. B.; Michnick, S. W.; Chiti, F.; Bai, Y.; Hagen, S. J.; Serrano, L.; Oliveberg, M.; Raleigh, D. P.; Wittung-Stafshede, P.; Radford, S. E.; Jackson, S. E.; Sosnick, T. R.; Marqusee, S.; Davidson, A.; Plaxco, K. W. Protein Sci. 2005, 14, 602. https://doi.org/10.1110/ps.041205405
  27. Park, S.-H. Bull. Korean Chem. Soc. 2009, 30, 1567 https://doi.org/10.5012/bkcs.2009.30.7.1567
  28. Baldwin, R. L. Fold. Des. 1996, 1, R1 https://doi.org/10.1016/S1359-0278(96)00003-X
  29. Wirmer, J.; Peti, W.; Schwalbe, H. J. Biomol. NMR 2006, 35, 175. https://doi.org/10.1007/s10858-006-9026-9
  30. Kim, P. S.; Baldwin, R. L. Annu. Rev. Biochem. 1982, 51, 459. https://doi.org/10.1146/annurev.bi.51.070182.002331
  31. Kim, P. S.; Baldwin, R. L. Annu. Rev. Biochem. 1990, 59, 631. https://doi.org/10.1146/annurev.bi.59.070190.003215
  32. Park, S.-H.; O'Neil, K. T.; Roder, H. Biochemistry 1997, 36, 14277. https://doi.org/10.1021/bi971914+

Cited by

  1. 소수성 상호작용이 HubWA 단백질의 폴딩 반응에 끼치는 영향 vol.63, pp.6, 2010, https://doi.org/10.5012/jkcs.2019.63.6.427