Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling Journal Article
| Authors: | Groß, M.; Robinson, C. V.; Mayhew, M.; Hartl, F. U.; Radford, S. E. |
| Article Title: | Significant hydrogen exchange protection in GroEL-bound DHFR is maintained during iterative rounds of substrate cycling |
| 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) hound 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 ATR. 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. |
| Keywords: | nonhuman; mass spectrometry; protein protein interaction; enzyme substrate; nucleotide sequence; substrate specificity; binding protein; ionization chamber; protein folding; dihydrofolate reductase; tetrahydrofolate dehydrogenase; cell protection; ligand binding; chaperone; hydrogen; citric acid cycle; molecular chaperones; respiratory chain; humans; priority journal; article; hydrogen exchange; substrate cycling |
| Journal Title: | Protein Science |
| Volume: | 5 |
| Issue: | 12 |
| ISSN: | 0961-8368 |
| Publisher: | Wiley Blackwell |
| Date Published: | 1996-12-01 |
| Start Page: | 2506 |
| End Page: | 2513 |
| Language: | English |
| PUBMED: | 8976559 |
| PROVIDER: | scopus |
| PMCID: | PMC2143321 |
| DOI: | 10.1002/pro.5560051213 |
| DOI/URL: | |
| Notes: | Article -- Export Date: 22 November 2017 -- Source: Scopus |
Altmetric
Citation Impact
BMJ Impact Analytics
Related MSK Work