Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling - PubMed (original) (raw)
Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling
M Gross et al. Protein Sci. 1996 Dec.
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.
Similar articles
- Conditions of forming protein complexes with GroEL can influence the mechanism of chaperonin-assisted refolding.
Gorovits BM, Horowitz PM. Gorovits BM, et al. J Biol Chem. 1997 Jan 3;272(1):32-5. doi: 10.1074/jbc.272.1.32. J Biol Chem. 1997. PMID: 8995221 - Native-like structure of a protein-folding intermediate bound to the chaperonin GroEL.
Goldberg MS, Zhang J, Sondek S, Matthews CR, Fox RO, Horwich AL. Goldberg MS, et al. Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1080-5. doi: 10.1073/pnas.94.4.1080. Proc Natl Acad Sci U S A. 1997. PMID: 9037009 Free PMC article. - GroEL-mediated folding of structurally homologous dihydrofolate reductases.
Clark AC, Frieden C. Clark AC, et al. J Mol Biol. 1997 May 2;268(2):512-25. doi: 10.1006/jmbi.1997.0969. J Mol Biol. 1997. PMID: 9159487 - Iterative annealing mechanism explains the functions of the GroEL and RNA chaperones.
Thirumalai D, Lorimer GH, Hyeon C. Thirumalai D, et al. Protein Sci. 2020 Feb;29(2):360-377. doi: 10.1002/pro.3795. Epub 2019 Dec 23. Protein Sci. 2020. PMID: 31800116 Free PMC article. Review. - Protein folding assisted by the GroEL/GroES chaperonin system.
Martin J. Martin J. Biochemistry (Mosc). 1998 Apr;63(4):374-81. Biochemistry (Mosc). 1998. PMID: 9556520 Review.
Cited by
- Folding trajectories of human dihydrofolate reductase inside the GroEL GroES chaperonin cavity and free in solution.
Horst R, Fenton WA, Englander SW, Wüthrich K, Horwich AL. Horst R, et al. Proc Natl Acad Sci U S A. 2007 Dec 26;104(52):20788-92. doi: 10.1073/pnas.0710042105. Epub 2007 Dec 19. Proc Natl Acad Sci U S A. 2007. PMID: 18093916 Free PMC article. - Scope and utility of hydrogen exchange as a tool for mapping landscapes.
Jaswal SS, Miranker AD. Jaswal SS, et al. Protein Sci. 2007 Nov;16(11):2378-90. doi: 10.1110/ps.072994207. Protein Sci. 2007. PMID: 17962401 Free PMC article. - Disulfide formation as a probe of folding in GroEL-GroES reveals correct formation of long-range bonds and editing of incorrect short-range ones.
Park ES, Fenton WA, Horwich AL. Park ES, et al. Proc Natl Acad Sci U S A. 2007 Feb 13;104(7):2145-50. doi: 10.1073/pnas.0610989104. Epub 2007 Feb 5. Proc Natl Acad Sci U S A. 2007. PMID: 17283341 Free PMC article. - GroEL-mediated protein folding: making the impossible, possible.
Lin Z, Rye HS. Lin Z, et al. Crit Rev Biochem Mol Biol. 2006 Jul-Aug;41(4):211-39. doi: 10.1080/10409230600760382. Crit Rev Biochem Mol Biol. 2006. PMID: 16849107 Free PMC article. Review. - Electron paramagnetic resonance and fluorescence studies of the conformation of aspartate aminotransferase bound to GroEL.
Berezov A, McNeill MJ, Iriarte A, Martinez-Carrion M. Berezov A, et al. Protein J. 2005 Nov;24(7-8):465-78. doi: 10.1007/s10930-005-7642-y. Protein J. 2005. PMID: 16328739
References
- FASEB J. 1996 Jan;10(1):93-101 - PubMed
- Biochemistry. 1992 Jan 14;31(1):218-29 - PubMed
- J Mol Biol. 1995 Jul 28;250(5):581-6 - PubMed
- Nature. 1996 Feb 1;379(6564):420-6 - PubMed
- Protein Sci. 1995 Feb;4(2):167-77 - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Research Materials