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
. 1997 May;6(5):1047–1056. doi: 10.1002/pro.5560060511

Hsc66 and Hsc20, a new heat shock cognate molecular chaperone system from Escherichia coli.

L E Vickery 1, J J Silberg 1, D T Ta 1
PMCID: PMC2143690  PMID: 9144776

Abstract

The hscA and hscB genes of Escherichia coli encode novel chaperone and co-chaperone proteins, designated Hsc66 and Hsc20, respectively. We have overproduced and purified Hsc66 and Hsc20 in high yield in E. coli and describe their initial characterization including absorbance, fluorescence, and circular dichroism spectra. Immunoblot analyses of E. coli cultures using antisera to Hsc66 and Hsc20 raised in rabbits establish that Hsc66 and Hsc20 are constitutively expressed at levels corresponding to cell concentration approximately 20 microM and approximately 10 microM, respectively. The levels do not change appreciably following heat shock (44 degrees C), but a small increase in Hsc20 is observed following a shift to 10 degrees C. Purified Hsc66 exhibits a low intrinsic ATPase activity (approximately 0.6 min-1 at 37 degrees C), and Hsc20 was found to stimulate this activity up to 3.8-fold with half-maximal stimulation at a concentration approximately 5 microM. These findings suggest that Hsc66 and Hsc20 comprise a molecular chaperone system similar to the prokaryotic DnaK/DnaJ and eukaryotic hsp70/hsp40 systems. Sequence differences between Hsc66 and Hsc20 compared to other members of this chaperone family, however, suggest that the Hsc66/Hsc20 system will display different peptide binding specificity and that it is likely to be subject to different regulatory mechanisms. The high level of constitutive expression and the lack of a major response to temperature changes suggest that Hsc66 and Hsc20 play an important cellular role(s) under non-stress conditions.

Full Text

The Full Text of this article is available as a PDF (2.9 MB).

Selected References

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

  1. Bardwell J. C., Craig E. A. Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous. Proc Natl Acad Sci U S A. 1984 Feb;81(3):848–852. doi: 10.1073/pnas.81.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Becker J., Craig E. A. Heat-shock proteins as molecular chaperones. Eur J Biochem. 1994 Jan 15;219(1-2):11–23. doi: 10.1007/978-3-642-79502-2_2. [DOI] [PubMed] [Google Scholar]
  3. Bukau B., Walker G. C. Cellular defects caused by deletion of the Escherichia coli dnaK gene indicate roles for heat shock protein in normal metabolism. J Bacteriol. 1989 May;171(5):2337–2346. doi: 10.1128/jb.171.5.2337-2346.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Caplan A. J., Cyr D. M., Douglas M. G. Eukaryotic homologues of Escherichia coli dnaJ: a diverse protein family that functions with hsp70 stress proteins. Mol Biol Cell. 1993 Jun;4(6):555–563. doi: 10.1091/mbc.4.6.555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  6. Cyr D. M., Langer T., Douglas M. G. DnaJ-like proteins: molecular chaperones and specific regulators of Hsp70. Trends Biochem Sci. 1994 Apr;19(4):176–181. doi: 10.1016/0968-0004(94)90281-x. [DOI] [PubMed] [Google Scholar]
  7. Flaherty K. M., DeLuca-Flaherty C., McKay D. B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature. 1990 Aug 16;346(6285):623–628. doi: 10.1038/346623a0. [DOI] [PubMed] [Google Scholar]
  8. Fleischmann R. D., Adams M. D., White O., Clayton R. A., Kirkness E. F., Kerlavage A. R., Bult C. J., Tomb J. F., Dougherty B. A., Merrick J. M. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science. 1995 Jul 28;269(5223):496–512. doi: 10.1126/science.7542800. [DOI] [PubMed] [Google Scholar]
  9. Flinta C., Persson B., Jörnvall H., von Heijne G. Sequence determinants of cytosolic N-terminal protein processing. Eur J Biochem. 1986 Jan 2;154(1):193–196. doi: 10.1111/j.1432-1033.1986.tb09378.x. [DOI] [PubMed] [Google Scholar]
  10. Gething M. J., Sambrook J. Protein folding in the cell. Nature. 1992 Jan 2;355(6355):33–45. doi: 10.1038/355033a0. [DOI] [PubMed] [Google Scholar]
  11. Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. doi: 10.1016/0003-2697(89)90602-7. [DOI] [PubMed] [Google Scholar]
  12. Hendrick J. P., Hartl F. U. The role of molecular chaperones in protein folding. FASEB J. 1995 Dec;9(15):1559–1569. doi: 10.1096/fasebj.9.15.8529835. [DOI] [PubMed] [Google Scholar]
  13. Herendeen S. L., VanBogelen R. A., Neidhardt F. C. Levels of major proteins of Escherichia coli during growth at different temperatures. J Bacteriol. 1979 Jul;139(1):185–194. doi: 10.1128/jb.139.1.185-194.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hill R. B., Flanagan J. M., Prestegard J. H. 1H and 15N magnetic resonance assignments, secondary structure, and tertiary fold of Escherichia coli DnaJ(1-78). Biochemistry. 1995 Apr 25;34(16):5587–5596. doi: 10.1021/bi00016a033. [DOI] [PubMed] [Google Scholar]
  15. Hirel P. H., Schmitter M. J., Dessen P., Fayat G., Blanquet S. Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8247–8251. doi: 10.1073/pnas.86.21.8247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Jordan R., McMacken R. Modulation of the ATPase activity of the molecular chaperone DnaK by peptides and the DnaJ and GrpE heat shock proteins. J Biol Chem. 1995 Mar 3;270(9):4563–4569. doi: 10.1074/jbc.270.9.4563. [DOI] [PubMed] [Google Scholar]
  17. Kamath-Loeb A. S., Lu C. Z., Suh W. C., Lonetto M. A., Gross C. A. Analysis of three DnaK mutant proteins suggests that progression through the ATPase cycle requires conformational changes. J Biol Chem. 1995 Dec 15;270(50):30051–30059. doi: 10.1074/jbc.270.50.30051. [DOI] [PubMed] [Google Scholar]
  18. Kawula T. H., Lelivelt M. J. Mutations in a gene encoding a new Hsp70 suppress rapid DNA inversion and bgl activation, but not proU derepression, in hns-1 mutant Escherichia coli. J Bacteriol. 1994 Feb;176(3):610–619. doi: 10.1128/jb.176.3.610-619.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  20. Lupas A. Coiled coils: new structures and new functions. Trends Biochem Sci. 1996 Oct;21(10):375–382. [PubMed] [Google Scholar]
  21. Mach H., Middaugh C. R., Lewis R. V. Statistical determination of the average values of the extinction coefficients of tryptophan and tyrosine in native proteins. Anal Biochem. 1992 Jan;200(1):74–80. doi: 10.1016/0003-2697(92)90279-g. [DOI] [PubMed] [Google Scholar]
  22. McCarty J. S., Buchberger A., Reinstein J., Bukau B. The role of ATP in the functional cycle of the DnaK chaperone system. J Mol Biol. 1995 May 26;249(1):126–137. doi: 10.1006/jmbi.1995.0284. [DOI] [PubMed] [Google Scholar]
  23. McCarty J. S., Walker G. C. DnaK as a thermometer: threonine-199 is site of autophosphorylation and is critical for ATPase activity. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9513–9517. doi: 10.1073/pnas.88.21.9513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. McKay D. B. Structure and mechanism of 70-kDa heat-shock-related proteins. Adv Protein Chem. 1993;44:67–98. doi: 10.1016/s0065-3233(08)60564-1. [DOI] [PubMed] [Google Scholar]
  25. O'Brien M. C., McKay D. B. How potassium affects the activity of the molecular chaperone Hsc70. I. Potassium is required for optimal ATPase activity. J Biol Chem. 1995 Feb 3;270(5):2247–2250. doi: 10.1074/jbc.270.5.2247. [DOI] [PubMed] [Google Scholar]
  26. Pace C. N., Vajdos F., Fee L., Grimsley G., Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995 Nov;4(11):2411–2423. doi: 10.1002/pro.5560041120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Palleros D. R., Shi L., Reid K. L., Fink A. L. Three-state denaturation of DnaK induced by guanidine hydrochloride. Evidence for an expandable intermediate. Biochemistry. 1993 Apr 27;32(16):4314–4321. doi: 10.1021/bi00067a021. [DOI] [PubMed] [Google Scholar]
  28. Park K., Flynn G. C., Rothman J. E., Fasman G. D. Conformational change of chaperone Hsc70 upon binding to a decapeptide: a circular dichroism study. Protein Sci. 1993 Mar;2(3):325–330. doi: 10.1002/pro.5560020304. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pellecchia M., Szyperski T., Wall D., Georgopoulos C., Wüthrich K. NMR structure of the J-domain and the Gly/Phe-rich region of the Escherichia coli DnaJ chaperone. J Mol Biol. 1996 Jul 12;260(2):236–250. doi: 10.1006/jmbi.1996.0395. [DOI] [PubMed] [Google Scholar]
  30. Rassow J., Voos W., Pfanner N. Partner proteins determine multiple functions of Hsp70. Trends Cell Biol. 1995 May;5(5):207–212. doi: 10.1016/s0962-8924(00)89001-7. [DOI] [PubMed] [Google Scholar]
  31. Sadis S., Raghavendra K., Hightower L. E. Secondary structure of the mammalian 70-kilodalton heat shock cognate protein analyzed by circular dichroism spectroscopy and secondary structure prediction. Biochemistry. 1990 Sep 11;29(36):8199–8206. doi: 10.1021/bi00488a001. [DOI] [PubMed] [Google Scholar]
  32. Seaton B. L., Vickery L. E. A gene encoding a DnaK/hsp70 homolog in Escherichia coli. Proc Natl Acad Sci U S A. 1994 Mar 15;91(6):2066–2070. doi: 10.1073/pnas.91.6.2066. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ta D. T., Vickery L. E. Cloning, sequencing, and overexpression of a [2Fe-2S] ferredoxin gene from Escherichia coli. J Biol Chem. 1992 Jun 5;267(16):11120–11125. [PubMed] [Google Scholar]
  34. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Welch W. J., Feramisco J. R. Rapid purification of mammalian 70,000-dalton stress proteins: affinity of the proteins for nucleotides. Mol Cell Biol. 1985 Jun;5(6):1229–1237. doi: 10.1128/mcb.5.6.1229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wilbanks S. M., Chen L., Tsuruta H., Hodgson K. O., McKay D. B. Solution small-angle X-ray scattering study of the molecular chaperone Hsc70 and its subfragments. Biochemistry. 1995 Sep 26;34(38):12095–12106. doi: 10.1021/bi00038a002. [DOI] [PubMed] [Google Scholar]
  37. Yang J. T., Wu C. S., Martinez H. M. Calculation of protein conformation from circular dichroism. Methods Enzymol. 1986;130:208–269. doi: 10.1016/0076-6879(86)30013-2. [DOI] [PubMed] [Google Scholar]
  38. Zhu X., Zhao X., Burkholder W. F., Gragerov A., Ogata C. M., Gottesman M. E., Hendrickson W. A. Structural analysis of substrate binding by the molecular chaperone DnaK. Science. 1996 Jun 14;272(5268):1606–1614. doi: 10.1126/science.272.5268.1606. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Ziegelhoffer T., Lopez-Buesa P., Craig E. A. The dissociation of ATP from hsp70 of Saccharomyces cerevisiae is stimulated by both Ydj1p and peptide substrates. J Biol Chem. 1995 May 5;270(18):10412–10419. doi: 10.1074/jbc.270.18.10412. [DOI] [PubMed] [Google Scholar]
  40. Zylicz M., Ang D., Georgopoulos C. The grpE protein of Escherichia coli. Purification and properties. J Biol Chem. 1987 Dec 25;262(36):17437–17442. [PubMed] [Google Scholar]
  41. Zylicz M., Georgopoulos C. Purification and properties of the Escherichia coli dnaK replication protein. J Biol Chem. 1984 Jul 25;259(14):8820–8825. [PubMed] [Google Scholar]

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

RESOURCES