Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1996 Dec;5(12):2506–2513. doi: 10.1002/pro.5560051213

Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling.

M Gross 1, C V Robinson 1, M Mayhew 1, F U Hartl 1, S E Radford 1
PMCID: PMC2143321  PMID: 8976559

Abstract

An unresolved key issue in the mechanism of protein folding assisted by the molecular chaperone GroEL is the nature of the substrate protein bound to the chaperonin at different stages of its reaction cycle. Here we describe the conformational properties of human dihydrofolate reductase (DHFR) bound to GroEL at different stages of its ATP-driven folding reaction, determined by hydrogen exchange labeling and electrospray ionization mass spectrometry. Considerable protection involving about 20 hydrogens is observed in DHFR bound to GroEL in the absence of ATP. Analysis of the line width of peaks in the mass spectra, together with fluorescence quenching and ANS binding studies, suggest that the bound DHFR is partially folded, but contains stable structure in a small region of the polypeptide chain. DHFR rebound to GroEL 3 min after initiating its folding by the addition of MgATP was also examined by hydrogen exchange, fluorescence quenching, and ANS binding. The results indicate that the extent of protection of the substrate protein rebound to GroEL is indistinguishable from that of the initial bound state. Despite this, small differences in the quenching coefficient and ANS binding properties are observed in the rebound state. On the basis of these results, we suggest that GroEL-assisted folding of DHFR occurs by minor structural adjustments to the partially folded substrate protein during iterative cycling, rather than by complete unfolding of this protein substrate on the chaperonin surface.

Full Text

The Full Text of this article is available as a PDF (2.3 MB).

Selected References

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

  1. Braig K., Simon M., Furuya F., Hainfeld J. F., Horwich A. L. A polypeptide bound by the chaperonin groEL is localized within a central cavity. Proc Natl Acad Sci U S A. 1993 May 1;90(9):3978–3982. doi: 10.1073/pnas.90.9.3978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Burston S. G., Ranson N. A., Clarke A. R. The origins and consequences of asymmetry in the chaperonin reaction cycle. J Mol Biol. 1995 May 26;249(1):138–152. doi: 10.1006/jmbi.1995.0285. [DOI] [PubMed] [Google Scholar]
  3. Chen S., Roseman A. M., Hunter A. S., Wood S. P., Burston S. G., Ranson N. A., Clarke A. R., Saibil H. R. Location of a folding protein and shape changes in GroEL-GroES complexes imaged by cryo-electron microscopy. Nature. 1994 Sep 15;371(6494):261–264. doi: 10.1038/371261a0. [DOI] [PubMed] [Google Scholar]
  4. Clark A. C., Hugo E., Frieden C. Determination of regions in the dihydrofolate reductase structure that interact with the molecular chaperonin GroEL. Biochemistry. 1996 May 7;35(18):5893–5901. doi: 10.1021/bi953051v. [DOI] [PubMed] [Google Scholar]
  5. Davies J. F., 2nd, Delcamp T. J., Prendergast N. J., Ashford V. A., Freisheim J. H., Kraut J. Crystal structures of recombinant human dihydrofolate reductase complexed with folate and 5-deazafolate. Biochemistry. 1990 Oct 9;29(40):9467–9479. doi: 10.1021/bi00492a021. [DOI] [PubMed] [Google Scholar]
  6. Eftink M. R., Ghiron C. A. Fluorescence quenching studies with proteins. Anal Biochem. 1981 Jul 1;114(2):199–227. doi: 10.1016/0003-2697(81)90474-7. [DOI] [PubMed] [Google Scholar]
  7. Hayer-Hartl M. K., Ewbank J. J., Creighton T. E., Hartl F. U. Conformational specificity of the chaperonin GroEL for the compact folding intermediates of alpha-lactalbumin. EMBO J. 1994 Jul 1;13(13):3192–3202. doi: 10.1002/j.1460-2075.1994.tb06618.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Horovitz A., Bochkareva E. S., Kovalenko O., Girshovich A. S. Mutation Ala2-->Ser destabilizes intersubunit interactions in the molecular chaperone GroEL. J Mol Biol. 1993 May 5;231(1):58–64. doi: 10.1006/jmbi.1993.1256. [DOI] [PubMed] [Google Scholar]
  9. Jackson G. S., Staniforth R. A., Halsall D. J., Atkinson T., Holbrook J. J., Clarke A. R., Burston S. G. Binding and hydrolysis of nucleotides in the chaperonin catalytic cycle: implications for the mechanism of assisted protein folding. Biochemistry. 1993 Mar 16;32(10):2554–2563. doi: 10.1021/bi00061a013. [DOI] [PubMed] [Google Scholar]
  10. Jennings P. A., Finn B. E., Jones B. E., Matthews C. R. A reexamination of the folding mechanism of dihydrofolate reductase from Escherichia coli: verification and refinement of a four-channel model. Biochemistry. 1993 Apr 13;32(14):3783–3789. doi: 10.1021/bi00065a034. [DOI] [PubMed] [Google Scholar]
  11. Jones B. E., Matthews C. R. Early intermediates in the folding of dihydrofolate reductase from Escherichia coli detected by hydrogen exchange and NMR. Protein Sci. 1995 Feb;4(2):167–177. doi: 10.1002/pro.5560040204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  13. Langer T., Lu C., Echols H., Flanagan J., Hayer M. K., Hartl F. U. Successive action of DnaK, DnaJ and GroEL along the pathway of chaperone-mediated protein folding. Nature. 1992 Apr 23;356(6371):683–689. doi: 10.1038/356683a0. [DOI] [PubMed] [Google Scholar]
  14. Lilie H., Buchner J. Interaction of GroEL with a highly structured folding intermediate: iterative binding cycles do not involve unfolding. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8100–8104. doi: 10.1073/pnas.92.18.8100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Martin J., Langer T., Boteva R., Schramel A., Horwich A. L., Hartl F. U. Chaperonin-mediated protein folding at the surface of groEL through a 'molten globule'-like intermediate. Nature. 1991 Jul 4;352(6330):36–42. doi: 10.1038/352036a0. [DOI] [PubMed] [Google Scholar]
  16. Martin J., Mayhew M., Langer T., Hartl F. U. The reaction cycle of GroEL and GroES in chaperonin-assisted protein folding. Nature. 1993 Nov 18;366(6452):228–233. doi: 10.1038/366228a0. [DOI] [PubMed] [Google Scholar]
  17. Mayhew M., da Silva A. C., Martin J., Erdjument-Bromage H., Tempst P., Hartl F. U. Protein folding in the central cavity of the GroEL-GroES chaperonin complex. Nature. 1996 Feb 1;379(6564):420–426. doi: 10.1038/379420a0. [DOI] [PubMed] [Google Scholar]
  18. Mendoza J. A., Horowitz P. M. Bound substrate polypeptides can generally stabilize the tetradecameric structure of Cpn60 and induce its reassembly from monomers. J Biol Chem. 1994 Oct 21;269(42):25963–25965. [PubMed] [Google Scholar]
  19. Merritt E. A., Murphy M. E. Raster3D Version 2.0. A program for photorealistic molecular graphics. Acta Crystallogr D Biol Crystallogr. 1994 Nov 1;50(Pt 6):869–873. doi: 10.1107/S0907444994006396. [DOI] [PubMed] [Google Scholar]
  20. Miranker A., Robinson C. V., Radford S. E., Aplin R. T., Dobson C. M. Detection of transient protein folding populations by mass spectrometry. Science. 1993 Nov 5;262(5135):896–900. doi: 10.1126/science.8235611. [DOI] [PubMed] [Google Scholar]
  21. Miranker A., Robinson C. V., Radford S. E., Dobson C. M. Investigation of protein folding by mass spectrometry. FASEB J. 1996 Jan;10(1):93–101. doi: 10.1096/fasebj.10.1.8566553. [DOI] [PubMed] [Google Scholar]
  22. Oefner C., D'Arcy A., Winkler F. K. Crystal structure of human dihydrofolate reductase complexed with folate. Eur J Biochem. 1988 Jun 1;174(2):377–385. doi: 10.1111/j.1432-1033.1988.tb14108.x. [DOI] [PubMed] [Google Scholar]
  23. Ranson N. A., Dunster N. J., Burston S. G., Clarke A. R. Chaperonins can catalyse the reversal of early aggregation steps when a protein misfolds. J Mol Biol. 1995 Jul 28;250(5):581–586. doi: 10.1006/jmbi.1995.0399. [DOI] [PubMed] [Google Scholar]
  24. Robinson C. V., Gross M., Eyles S. J., Ewbank J. J., Mayhew M., Hartl F. U., Dobson C. M., Radford S. E. Conformation of GroEL-bound alpha-lactalbumin probed by mass spectrometry. Nature. 1994 Dec 15;372(6507):646–651. doi: 10.1038/372646a0. [DOI] [PubMed] [Google Scholar]
  25. Rospert S., Looser R., Dubaquie Y., Matouschek A., Glick B. S., Schatz G. Hsp60-independent protein folding in the matrix of yeast mitochondria. EMBO J. 1996 Feb 15;15(4):764–774. [PMC free article] [PubMed] [Google Scholar]
  26. Schweitzer B. I., Srimatkandada S., Gritsman H., Sheridan R., Venkataraghavan R., Bertino J. R. Probing the role of two hydrophobic active site residues in the human dihydrofolate reductase by site-directed mutagenesis. J Biol Chem. 1989 Dec 5;264(34):20786–20795. [PubMed] [Google Scholar]
  27. Stockman B. J., Nirmala N. R., Wagner G., Delcamp T. J., DeYarman M. T., Freisheim J. H. Methotrexate binds in a non-productive orientation to human dihydrofolate reductase in solution, based on NMR spectroscopy. FEBS Lett. 1991 Jun 3;283(2):267–269. doi: 10.1016/0014-5793(91)80604-2. [DOI] [PubMed] [Google Scholar]
  28. Stockman B. J., Nirmala N. R., Wagner G., Delcamp T. J., DeYarman M. T., Freisheim J. H. Sequence-specific 1H and 15N resonance assignments for human dihydrofolate reductase in solution. Biochemistry. 1992 Jan 14;31(1):218–229. doi: 10.1021/bi00116a031. [DOI] [PubMed] [Google Scholar]
  29. Thiyagarajan P., Henderson S. J., Joachimiak A. Solution structures of GroEL and its complex with rhodanese from small-angle neutron scattering. Structure. 1996 Jan 15;4(1):79–88. doi: 10.1016/s0969-2126(96)00011-1. [DOI] [PubMed] [Google Scholar]
  30. Todd M. J., Viitanen P. V., Lorimer G. H. Dynamics of the chaperonin ATPase cycle: implications for facilitated protein folding. Science. 1994 Jul 29;265(5172):659–666. doi: 10.1126/science.7913555. [DOI] [PubMed] [Google Scholar]
  31. Weissman J. S., Kashi Y., Fenton W. A., Horwich A. L. GroEL-mediated protein folding proceeds by multiple rounds of binding and release of nonnative forms. Cell. 1994 Aug 26;78(4):693–702. doi: 10.1016/0092-8674(94)90533-9. [DOI] [PubMed] [Google Scholar]
  32. Weissman J. S., Rye H. S., Fenton W. A., Beechem J. M., Horwich A. L. Characterization of the active intermediate of a GroEL-GroES-mediated protein folding reaction. Cell. 1996 Feb 9;84(3):481–490. doi: 10.1016/s0092-8674(00)81293-3. [DOI] [PubMed] [Google Scholar]
  33. Yifrach O., Horovitz A. Allosteric control by ATP of non-folded protein binding to GroEL. J Mol Biol. 1996 Jan 26;255(3):356–361. doi: 10.1006/jmbi.1996.0028. [DOI] [PubMed] [Google Scholar]
  34. Zahn R., Perrett S., Stenberg G., Fersht A. R. Catalysis of amide proton exchange by the molecular chaperones GroEL and SecB. Science. 1996 Feb 2;271(5249):642–645. doi: 10.1126/science.271.5249.642. [DOI] [PubMed] [Google Scholar]
  35. Zahn R., Spitzfaden C., Ottiger M., Wüthrich K., Plückthun A. Destabilization of the complete protein secondary structure on binding to the chaperone GroEL. Nature. 1994 Mar 17;368(6468):261–265. doi: 10.1038/368261a0. [DOI] [PubMed] [Google Scholar]

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

RESOURCES