Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 Jun;8(6):1276–1285. doi: 10.1110/ps.8.6.1276

Probing the role of water in the tryptophan repressor-operator complex.

M P Brown 1, A O Grillo 1, M Boyer 1, C A Royer 1
PMCID: PMC2144343  PMID: 10386877

Abstract

The Escherichia coli tryptophan repressor protein (TR) represses the transcription of several genes in response to the concentration of tryptophan in the environment. In the co-crystal structure of TR bound to a DNA fragment containing its target very few direct contacts between TR and the DNA were observed. In contrast, a number of solvent mediated contacts were apparent. NMR solution structures, however, did not resolve any solvent mediated bonds at the complex interface. To probe for the role of water in TR operator recognition, the effect of osmolytes on the interactions between TR and a target oligonucleotide bearing the operator site was examined. In the absence of specific solvent mediated hydrogen bonding interactions between the protein and the DNA, increasing osmolyte concentration is expected to strongly stabilize the TR operator interaction due to the large amount of macromolecular surface area buried upon complexation. The results of our studies indicate that xylose did not alter the binding affinity significantly, while glycerol and PEG had a small stabilizing effect. A study of binding as a function of betaine concentration revealed that this osmolyte at low concentration results in a stabilization of the 1:1 TR/operator complex, but at higher concentrations leads to a switching between binding modes to favor tandem binding. Analysis of the effects of betaine on the 1:1 complex suggest that this osmolyte has about 78% of the expected effect. If one accepts the analysis in terms of the number of water molecules excluded upon complexation, these results suggest that about 75 water molecules remain at the interface of the 1:1 dimer/DNA complex. This value is consistent with the number of water molecules found at the interface in the crystallographically determined structure and supports the notion that interfacial waters play an important thermodynamic role in the specific complexation of one TR dimer with its target DNA. However, the complexity of the effects of betaine and the small or negligible effects of the other osmolytes could also arise from osmolyte induced competition between antagonistic coupled reactions.

Full Text

The Full Text of this article is available as a PDF (444.0 KB).

Selected References

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

  1. Arakawa T., Timasheff S. N. The stabilization of proteins by osmolytes. Biophys J. 1985 Mar;47(3):411–414. doi: 10.1016/S0006-3495(85)83932-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bass S., Sugiono P., Arvidson D. N., Gunsalus R. P., Youderian P. DNA specificity determinants of Escherichia coli tryptophan repressor binding. Genes Dev. 1987 Aug;1(6):565–572. doi: 10.1101/gad.1.6.565. [DOI] [PubMed] [Google Scholar]
  3. Carey J., Lewis D. E., Lavoie T. A., Yang J. How does trp repressor bind to its operator? J Biol Chem. 1991 Dec 25;266(36):24509–24513. [PubMed] [Google Scholar]
  4. Colombo M. F., Rau D. C., Parsegian V. A. Protein solvation in allosteric regulation: a water effect on hemoglobin. Science. 1992 May 1;256(5057):655–659. doi: 10.1126/science.1585178. [DOI] [PubMed] [Google Scholar]
  5. Fernando T., Royer C. Role of protein--protein interactions in the regulation of transcription by trp repressor investigated by fluorescence spectroscopy. Biochemistry. 1992 Apr 7;31(13):3429–3441. doi: 10.1021/bi00128a018. [DOI] [PubMed] [Google Scholar]
  6. Garner M. M., Rau D. C. Water release associated with specific binding of gal repressor. EMBO J. 1995 Mar 15;14(6):1257–1263. doi: 10.1002/j.1460-2075.1995.tb07109.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Grillo A. O., Brown M. P., Royer C. A. Probing the physical basis for trp repressor-operator recognition. J Mol Biol. 1999 Apr 2;287(3):539–554. doi: 10.1006/jmbi.1999.2625. [DOI] [PubMed] [Google Scholar]
  8. Gunsalus R. P., Yanofsky C. Nucleotide sequence and expression of Escherichia coli trpR, the structural gene for the trp aporepressor. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7117–7121. doi: 10.1073/pnas.77.12.7117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Haran T. E., Joachimiak A., Sigler P. B. The DNA target of the trp repressor. EMBO J. 1992 Aug;11(8):3021–3030. doi: 10.1002/j.1460-2075.1992.tb05372.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Joachimiak A., Haran T. E., Sigler P. B. Mutagenesis supports water mediated recognition in the trp repressor-operator system. EMBO J. 1994 Jan 15;13(2):367–372. doi: 10.1002/j.1460-2075.1994.tb06270.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Joachimiak A., Kelley R. L., Gunsalus R. P., Yanofsky C., Sigler P. B. Purification and characterization of trp aporepressor. Proc Natl Acad Sci U S A. 1983 Feb;80(3):668–672. doi: 10.1073/pnas.80.3.668. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kelley R. L., Yanofsky C. Mutational studies with the trp repressor of Escherichia coli support the helix-turn-helix model of repressor recognition of operator DNA. Proc Natl Acad Sci U S A. 1985 Jan;82(2):483–487. doi: 10.1073/pnas.82.2.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Kornblatt J. A., Hoa G. H. A nontraditional role for water in the cytochrome c oxidase reaction. Biochemistry. 1990 Oct 9;29(40):9370–9376. doi: 10.1021/bi00492a010. [DOI] [PubMed] [Google Scholar]
  14. Lawson C. L., Carey J. Tandem binding in crystals of a trp repressor/operator half-site complex. Nature. 1993 Nov 11;366(6451):178–182. doi: 10.1038/366178a0. [DOI] [PubMed] [Google Scholar]
  15. LeTilly V., Royer C. A. Fluorescence anisotropy assays implicate protein-protein interactions in regulating trp repressor DNA binding. Biochemistry. 1993 Aug 3;32(30):7753–7758. doi: 10.1021/bi00081a021. [DOI] [PubMed] [Google Scholar]
  16. Liu Y. C., Matthews K. S. Dependence of trp repressor-operator affinity, stoichiometry, and apparent cooperativity on DNA sequence and size. J Biol Chem. 1993 Nov 5;268(31):23239–23249. [PubMed] [Google Scholar]
  17. Morton C. J., Ladbury J. E. Water-mediated protein-DNA interactions: the relationship of thermodynamics to structural detail. Protein Sci. 1996 Oct;5(10):2115–2118. doi: 10.1002/pro.5560051018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Otwinowski Z., Schevitz R. W., Zhang R. G., Lawson C. L., Joachimiak A., Marmorstein R. Q., Luisi B. F., Sigler P. B. Crystal structure of trp repressor/operator complex at atomic resolution. Nature. 1988 Sep 22;335(6188):321–329. doi: 10.1038/335321a0. [DOI] [PubMed] [Google Scholar]
  19. Parsegian V. A., Rand R. P., Rau D. C. Macromolecules and water: probing with osmotic stress. Methods Enzymol. 1995;259:43–94. doi: 10.1016/0076-6879(95)59039-0. [DOI] [PubMed] [Google Scholar]
  20. Prouty M. S., Schechter A. N., Parsegian V. A. Chemical potential measurements of deoxyhemoglobin S polymerization. Determination of the phase diagram of an assembling protein. J Mol Biol. 1985 Aug 5;184(3):517–528. doi: 10.1016/0022-2836(85)90298-0. [DOI] [PubMed] [Google Scholar]
  21. Rand R. P., Fuller N. L., Butko P., Francis G., Nicholls P. Measured change in protein solvation with substrate binding and turnover. Biochemistry. 1993 Jun 15;32(23):5925–5929. doi: 10.1021/bi00074a001. [DOI] [PubMed] [Google Scholar]
  22. Reedstrom R. J., Brown M. P., Grillo A., Roen D., Royer C. A. Affinity and specificity of trp repressor-DNA interactions studied with fluorescent oligonucleotides. J Mol Biol. 1997 Oct 31;273(3):572–585. doi: 10.1006/jmbi.1997.1333. [DOI] [PubMed] [Google Scholar]
  23. Reid C., Rand R. P. Probing protein hydration and conformational states in solution. Biophys J. 1997 Mar;72(3):1022–1030. doi: 10.1016/S0006-3495(97)78754-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Robinson C. R., Sligar S. G. Changes in solvation during DNA binding and cleavage are critical to altered specificity of the EcoRI endonuclease. Proc Natl Acad Sci U S A. 1998 Mar 3;95(5):2186–2191. doi: 10.1073/pnas.95.5.2186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Robinson C. R., Sligar S. G. Heterogeneity in molecular recognition by restriction endonucleases: osmotic and hydrostatic pressure effects on BamHI, Pvu II, and EcoRV specificity. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3444–3448. doi: 10.1073/pnas.92.8.3444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Robinson C. R., Sligar S. G. Hydrostatic and osmotic pressure as tools to study macromolecular recognition. Methods Enzymol. 1995;259:395–427. doi: 10.1016/0076-6879(95)59054-4. [DOI] [PubMed] [Google Scholar]
  27. Robinson C. R., Sligar S. G. Hydrostatic pressure reverses osmotic pressure effects on the specificity of EcoRI-DNA interactions. Biochemistry. 1994 Apr 5;33(13):3787–3793. doi: 10.1021/bi00179a001. [DOI] [PubMed] [Google Scholar]
  28. Robinson C. R., Sligar S. G. Molecular recognition mediated by bound water. A mechanism for star activity of the restriction endonuclease EcoRI. J Mol Biol. 1993 Nov 20;234(2):302–306. doi: 10.1006/jmbi.1993.1586. [DOI] [PubMed] [Google Scholar]
  29. Robinson C. R., Sligar S. G. Participation of water in Hin recombinase--DNA recognition. Protein Sci. 1996 Oct;5(10):2119–2124. doi: 10.1002/pro.5560051019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rose J. K., Yanofsky C. Interaction of the operator of the tryptophan operon with repressor. Proc Natl Acad Sci U S A. 1974 Aug;71(8):3134–3138. doi: 10.1073/pnas.71.8.3134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Royer C. A., Beechem J. M. Numerical analysis of binding data: advantages, practical aspects, and implications. Methods Enzymol. 1992;210:481–505. doi: 10.1016/0076-6879(92)10025-9. [DOI] [PubMed] [Google Scholar]
  32. Royer C. A. Improvements in the numerical analysis of thermodynamic data from biomolecular complexes. Anal Biochem. 1993 Apr;210(1):91–97. doi: 10.1006/abio.1993.1155. [DOI] [PubMed] [Google Scholar]
  33. Royer C. A., Smith W. R., Beechem J. M. Analysis of binding in macromolecular complexes: a generalized numerical approach. Anal Biochem. 1990 Dec;191(2):287–294. doi: 10.1016/0003-2697(90)90221-t. [DOI] [PubMed] [Google Scholar]
  34. Schevitz R. W., Otwinowski Z., Joachimiak A., Lawson C. L., Sigler P. B. The three-dimensional structure of trp repressor. 1985 Oct 31-Nov 6Nature. 317(6040):782–786. doi: 10.1038/317782a0. [DOI] [PubMed] [Google Scholar]
  35. Schwabe J. W. The role of water in protein-DNA interactions. Curr Opin Struct Biol. 1997 Feb;7(1):126–134. doi: 10.1016/s0959-440x(97)80016-4. [DOI] [PubMed] [Google Scholar]
  36. Shakked Z., Guzikevich-Guerstein G., Frolow F., Rabinovich D., Joachimiak A., Sigler P. B. Determinants of repressor/operator recognition from the structure of the trp operator binding site. Nature. 1994 Mar 31;368(6470):469–473. doi: 10.1038/368469a0. [DOI] [PubMed] [Google Scholar]
  37. Timasheff S. N. In disperse solution, "osmotic stress" is a restricted case of preferential interactions. Proc Natl Acad Sci U S A. 1998 Jun 23;95(13):7363–7367. doi: 10.1073/pnas.95.13.7363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Timasheff S. N. The control of protein stability and association by weak interactions with water: how do solvents affect these processes? Annu Rev Biophys Biomol Struct. 1993;22:67–97. doi: 10.1146/annurev.bb.22.060193.000435. [DOI] [PubMed] [Google Scholar]
  39. Vossen K. M., Wolz R., Daugherty M. A., Fried M. G. Role of macromolecular hydration in the binding of the Escherichia coli cyclic AMP receptor to DNA. Biochemistry. 1997 Sep 30;36(39):11640–11647. doi: 10.1021/bi971193e. [DOI] [PubMed] [Google Scholar]
  40. Yang J., Gunasekera A., Lavoie T. A., Jin L., Lewis D. E., Carey J. In vivo and in vitro studies of TrpR-DNA interactions. J Mol Biol. 1996 Apr 26;258(1):37–52. doi: 10.1006/jmbi.1996.0232. [DOI] [PubMed] [Google Scholar]
  41. Zhang H., Zhao D., Revington M., Lee W., Jia X., Arrowsmith C., Jardetzky O. The solution structures of the trp repressor-operator DNA complex. J Mol Biol. 1994 May 13;238(4):592–614. doi: 10.1006/jmbi.1994.1317. [DOI] [PubMed] [Google Scholar]
  42. Zurawski G., Gunsalus R. P., Brown K. D., Yanofsky C. Structure and regulation of aroH, the structural gene for the tryptophan-repressible 3-deoxy-D-arabino-heptulosonic acid-7-phosphate synthetase of Escherichia coli. J Mol Biol. 1981 Jan 5;145(1):47–73. doi: 10.1016/0022-2836(81)90334-x. [DOI] [PubMed] [Google Scholar]
  43. von Hippel P. H., Berg O. G. On the specificity of DNA-protein interactions. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1608–1612. doi: 10.1073/pnas.83.6.1608. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES