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. 2000 Feb;9(2):344–352. doi: 10.1110/ps.9.2.344

Proline inhibits aggregation during protein refolding.

D Samuel 1, T K Kumar 1, G Ganesh 1, G Jayaraman 1, P W Yang 1, M M Chang 1, V D Trivedi 1, S L Wang 1, K C Hwang 1, D K Chang 1, C Yu 1
PMCID: PMC2144545  PMID: 10716186

Abstract

The in vitro refolding of hen egg-white lysozyme is studied in the presence of various osmolytes. Proline is found to prevent aggregation during protein refolding. However, other osmolytes used in this study fail to exhibit a similar property. Experimental evidence suggests that proline inhibits protein aggregation by binding to folding intermediate(s) and trapping the folding intermediate(s) into enzymatically inactive, "aggregation-insensitive" state(s). However, elimination of proline from the refolded protein mixture results in significant recovery of the bacteriolytic activity. At higher concentrations (>1.5 M), proline is shown to form loose, higher-order molecular aggregate(s). The supramolecular assembly of proline is found to possess an amphipathic character. Formation of higher-order aggregates is believed to be crucial for proline to function as a protein folding aid. In addition to its role in osmoregulation under water stress conditions, the results of this study hint at the possibility of proline behaving as a protein folding chaperone.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brown A. D., Simpson J. R. Water relations of sugar-tolerant yeasts: the role of intracellular polyols. J Gen Microbiol. 1972 Oct;72(3):589–591. doi: 10.1099/00221287-72-3-589. [DOI] [PubMed] [Google Scholar]
  2. Cleland J. L., Wang D. I. Refolding and aggregation of bovine carbonic anhydrase B: quasi-elastic light scattering analysis. Biochemistry. 1990 Dec 18;29(50):11072–11078. doi: 10.1021/bi00502a009. [DOI] [PubMed] [Google Scholar]
  3. Dill K. A. Dominant forces in protein folding. Biochemistry. 1990 Aug 7;29(31):7133–7155. doi: 10.1021/bi00483a001. [DOI] [PubMed] [Google Scholar]
  4. Goldberg M. E., Rudolph R., Jaenicke R. A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme. Biochemistry. 1991 Mar 19;30(11):2790–2797. doi: 10.1021/bi00225a008. [DOI] [PubMed] [Google Scholar]
  5. Karuppiah N., Sharma A. Cyclodextrins as protein folding aids. Biochem Biophys Res Commun. 1995 Jun 6;211(1):60–66. doi: 10.1006/bbrc.1995.1778. [DOI] [PubMed] [Google Scholar]
  6. Kirk W. R., Kurian E., Prendergast F. G. Characterization of the sources of protein-ligand affinity: 1-sulfonato-8-(1')anilinonaphthalene binding to intestinal fatty acid binding protein. Biophys J. 1996 Jan;70(1):69–83. doi: 10.1016/S0006-3495(96)79592-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kumar T. K., Gopalakrishna K., Ramakrishna T., Pandit M. W. Refolding of RNAse A at high concentrations: identification of non-native species. Int J Biol Macromol. 1994 Aug;16(4):171–176. doi: 10.1016/0141-8130(94)90047-7. [DOI] [PubMed] [Google Scholar]
  8. Kumar T. K., Jayaraman G., Lee C. S., Sivaraman T., Lin W. Y., Yu C. Identification of 'molten globule'-like state in all beta-sheet protein. Biochem Biophys Res Commun. 1995 Feb 15;207(2):536–543. doi: 10.1006/bbrc.1995.1221. [DOI] [PubMed] [Google Scholar]
  9. Kumar T. K., Yang P. W., Lin S. H., Wu C. Y., Lei B., Lo S. J., Tu S. C., Yu C. Cloning, direct expression, and purification of a snake venom cardiotoxin in Escherichia coli. Biochem Biophys Res Commun. 1996 Feb 15;219(2):450–456. doi: 10.1006/bbrc.1996.0254. [DOI] [PubMed] [Google Scholar]
  10. Lin T. Y., Timasheff S. N. Why do some organisms use a urea-methylamine mixture as osmolyte? Thermodynamic compensation of urea and trimethylamine N-oxide interactions with protein. Biochemistry. 1994 Oct 25;33(42):12695–12701. doi: 10.1021/bi00208a021. [DOI] [PubMed] [Google Scholar]
  11. Liu Y., Bolen D. W. The peptide backbone plays a dominant role in protein stabilization by naturally occurring osmolytes. Biochemistry. 1995 Oct 3;34(39):12884–12891. doi: 10.1021/bi00039a051. [DOI] [PubMed] [Google Scholar]
  12. Marston F. A. The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem J. 1986 Nov 15;240(1):1–12. doi: 10.1042/bj2400001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Matulis D., Lovrien R. 1-Anilino-8-naphthalene sulfonate anion-protein binding depends primarily on ion pair formation. Biophys J. 1998 Jan;74(1):422–429. doi: 10.1016/S0006-3495(98)77799-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Orsini G., Goldberg M. E. The renaturation of reduced chymotrypsinogen A in guanidine HCl. Refolding versus aggregation. J Biol Chem. 1978 May 25;253(10):3453–3458. [PubMed] [Google Scholar]
  15. Orsini G., Skrzynia C., Goldberg M. E. The renaturation of reduced polyalanyl-chymotrypsinogen and chymotrypsinogen. Eur J Biochem. 1975 Nov 15;59(2):433–440. doi: 10.1111/j.1432-1033.1975.tb02471.x. [DOI] [PubMed] [Google Scholar]
  16. Ptitsyn O. B., Pain R. H., Semisotnov G. V., Zerovnik E., Razgulyaev O. I. Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 1990 Mar 12;262(1):20–24. doi: 10.1016/0014-5793(90)80143-7. [DOI] [PubMed] [Google Scholar]
  17. Rozema D., Gellman S. H. Artificial chaperone-assisted refolding of carbonic anhydrase B. J Biol Chem. 1996 Feb 16;271(7):3478–3487. doi: 10.1074/jbc.271.7.3478. [DOI] [PubMed] [Google Scholar]
  18. Rozema D., Gellman S. H. Artificial chaperone-assisted refolding of denatured-reduced lysozyme: modulation of the competition between renaturation and aggregation. Biochemistry. 1996 Dec 10;35(49):15760–15771. doi: 10.1021/bi961638j. [DOI] [PubMed] [Google Scholar]
  19. Samuel D., Kumar T. K., Jayaraman G., Yang P. W., Yu C. Proline is a protein solubilizing solute. Biochem Mol Biol Int. 1997 Feb;41(2):235–242. doi: 10.1080/15216549700201241. [DOI] [PubMed] [Google Scholar]
  20. Saxena V. P., Wetlaufer D. B. Formation of three-dimensional structure in proteins. I. Rapid nonenzymic reactivation of reduced lysozyme. Biochemistry. 1970 Dec 8;9(25):5015–5023. doi: 10.1021/bi00827a028. [DOI] [PubMed] [Google Scholar]
  21. Schobert B., Tschesche H. Unusual solution properties of proline and its interaction with proteins. Biochim Biophys Acta. 1978 Jun 15;541(2):270–277. doi: 10.1016/0304-4165(78)90400-2. [DOI] [PubMed] [Google Scholar]
  22. Semisotnov G. V., Uversky V. N., Sokolovsky I. V., Gutin A. M., Razgulyaev O. I., Rodionova N. A. Two slow stages in refolding of bovine carbonic anhydrase B are due to proline isomerization. J Mol Biol. 1990 Jun 5;213(3):561–568. doi: 10.1016/S0022-2836(05)80215-3. [DOI] [PubMed] [Google Scholar]
  23. Sivaraman T., Kumar T. K., Jayaraman G., Han C. C., Yu C. Characterization of a partially structured state in an all-beta-sheet protein. Biochem J. 1997 Jan 15;321(Pt 2):457–464. doi: 10.1042/bj3210457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Sivaraman T., Kumar T. K., Jayaraman G., Yu C. The mechanism of 2,2,2-trichloroacetic acid-induced protein precipitation. J Protein Chem. 1997 May;16(4):291–297. doi: 10.1023/a:1026357009886. [DOI] [PubMed] [Google Scholar]
  25. Tandon S., Horowitz P. Detergent-assisted refolding of guanidinium chloride-denatured rhodanese. The effect of lauryl maltoside. J Biol Chem. 1986 Nov 25;261(33):15615–15618. [PubMed] [Google Scholar]
  26. Thomas P. J., Qu B. H., Pedersen P. L. Defective protein folding as a basis of human disease. Trends Biochem Sci. 1995 Nov;20(11):456–459. doi: 10.1016/s0968-0004(00)89100-8. [DOI] [PubMed] [Google Scholar]
  27. Wang A., Bolen D. W. A naturally occurring protective system in urea-rich cells: mechanism of osmolyte protection of proteins against urea denaturation. Biochemistry. 1997 Jul 29;36(30):9101–9108. doi: 10.1021/bi970247h. [DOI] [PubMed] [Google Scholar]
  28. Wang A., Bolen D. W. Effect of proline on lactate dehydrogenase activity: testing the generality and scope of the compatibility paradigm. Biophys J. 1996 Oct;71(4):2117–2122. doi: 10.1016/S0006-3495(96)79410-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wetlaufer D. B., Xie Y. Control of aggregation in protein refolding: a variety of surfactants promote renaturation of carbonic anhydrase II. Protein Sci. 1995 Aug;4(8):1535–1543. doi: 10.1002/pro.5560040811. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wetzel R. Mutations and off-pathway aggregation of proteins. Trends Biotechnol. 1994 May;12(5):193–198. doi: 10.1016/0167-7799(94)90082-5. [DOI] [PubMed] [Google Scholar]
  31. Yancey P. H., Clark M. E., Hand S. C., Bowlus R. D., Somero G. N. Living with water stress: evolution of osmolyte systems. Science. 1982 Sep 24;217(4566):1214–1222. doi: 10.1126/science.7112124. [DOI] [PubMed] [Google Scholar]
  32. Zardeneta G., Horowitz P. M. Micelle-assisted protein folding. Denatured rhodanese binding to cardiolipin-containing lauryl maltoside micelles results in slower refolding kinetics but greater enzyme reactivation. J Biol Chem. 1992 Mar 25;267(9):5811–5816. [PubMed] [Google Scholar]
  33. Zettlmeissl G., Rudolph R., Jaenicke R. Reconstitution of lactic dehydrogenase. Noncovalent aggregation vs. reactivation. 1. Physical properties and kinetics of aggregation. Biochemistry. 1979 Dec 11;18(25):5567–5571. doi: 10.1021/bi00592a007. [DOI] [PubMed] [Google Scholar]

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