TY - JOUR
T1 - The chaperonin GroEL binds to late-folding non-native conformations present in native Escherichia coli and murine dihydrofolate reductases
AU - Clark, A. Clay
AU - Frieden, Carl
N1 - Funding Information:
We thank Drs Brad Karon, Sydney Hoeltzli, Jenny Buzan, George Drysdale, Keeyhuk Kim, and Linda Kurz for helpful discussions. This work was supported by National Institutes of Health grant DK13332
PY - 1999/1/29
Y1 - 1999/1/29
N2 - Dihydrofolate reductases from mouse (MuDHFR) or Escherichia coli (EcDHFR) are shown to refold via several intermediate forms, each of which can bind to the chaperonin GroEL. When stable complexes with GroEL are formed, they consist of late-folding intermediates. In addition, we find that late-folding intermediates that are present in the native enzyme bind to GroEL. For the E. coli and murine proteins, the extent of protein bound increases as the temperature is increased from 8°C to 42°C, at which temperature either protein is completely bound as the last (EcDHFR) or the last two (MuDHFR) folding intermediate(s). Thus for EcDHFR, the binding is transient at low temperature (< 30°C) and stable at high temperature (> 35°C). For MuDHFR, complex formation appears less temperature dependent. In general, the data demonstrate that the overall binding free energy for the interaction of GroEL with native DHFR is the sum of the free energy for the first step in DHFR unfolding, which is unfavorable, and the free energy of binding the non-native conformation, which is favorable. For EcDHFR, this results in an overall binding free energy that is unfavorable below 30°C. Over the temperature range of 8°C to 42°C, GroEL binds MuDHFR more tightly than EcDHFR, due partially to a small free energy difference between two pre-existing non-native conformations of MuDHFR, resulting in binding to more than one folding intermediate.
AB - Dihydrofolate reductases from mouse (MuDHFR) or Escherichia coli (EcDHFR) are shown to refold via several intermediate forms, each of which can bind to the chaperonin GroEL. When stable complexes with GroEL are formed, they consist of late-folding intermediates. In addition, we find that late-folding intermediates that are present in the native enzyme bind to GroEL. For the E. coli and murine proteins, the extent of protein bound increases as the temperature is increased from 8°C to 42°C, at which temperature either protein is completely bound as the last (EcDHFR) or the last two (MuDHFR) folding intermediate(s). Thus for EcDHFR, the binding is transient at low temperature (< 30°C) and stable at high temperature (> 35°C). For MuDHFR, complex formation appears less temperature dependent. In general, the data demonstrate that the overall binding free energy for the interaction of GroEL with native DHFR is the sum of the free energy for the first step in DHFR unfolding, which is unfavorable, and the free energy of binding the non-native conformation, which is favorable. For EcDHFR, this results in an overall binding free energy that is unfavorable below 30°C. Over the temperature range of 8°C to 42°C, GroEL binds MuDHFR more tightly than EcDHFR, due partially to a small free energy difference between two pre-existing non-native conformations of MuDHFR, resulting in binding to more than one folding intermediate.
KW - Binding free energy
KW - Chaperonin
KW - DHFR
KW - Kinetics
KW - Protein folding
UR - http://www.scopus.com/inward/record.url?scp=0033613819&partnerID=8YFLogxK
U2 - 10.1006/jmbi.1998.2403
DO - 10.1006/jmbi.1998.2403
M3 - Article
C2 - 9917411
AN - SCOPUS:0033613819
SN - 0022-2836
VL - 285
SP - 1777
EP - 1788
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -