TY - JOUR
T1 - Native Escherichia coli and murine dihydrofolate reductases contain late-folding non-native structures
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 - We have examined the equilibrium and kinetic folding properties of two structurally homologous dihydrofolate reductases, Escherichia coli DHFR (EcDHFR) and murine DHFR (MuDHFR), as a function of temperature and ligand concentration. Conformational heterogeneity in native DHFR is well documented, and the results demonstrate that the non-native form(s) represents late intermediate(s) in the folding process. We have measured the concentrations of native and non-native forms and the rate constants for their interconversion over a temperature range of 3°C to 49°C, allowing characterization of the thermodynamic as well as the kinetic properties of the final folding step(s) relative to the overall folding reaction. Differences in ligand binding suggest that the intermediate structures for these two proteins may be different during refolding.
AB - We have examined the equilibrium and kinetic folding properties of two structurally homologous dihydrofolate reductases, Escherichia coli DHFR (EcDHFR) and murine DHFR (MuDHFR), as a function of temperature and ligand concentration. Conformational heterogeneity in native DHFR is well documented, and the results demonstrate that the non-native form(s) represents late intermediate(s) in the folding process. We have measured the concentrations of native and non-native forms and the rate constants for their interconversion over a temperature range of 3°C to 49°C, allowing characterization of the thermodynamic as well as the kinetic properties of the final folding step(s) relative to the overall folding reaction. Differences in ligand binding suggest that the intermediate structures for these two proteins may be different during refolding.
KW - Conformational heterogeneity
KW - DHFR
KW - Equilibrium folding/unfolding
KW - Kinetics
KW - Stopped-flow fluorescence
UR - http://www.scopus.com/inward/record.url?scp=0033613814&partnerID=8YFLogxK
U2 - 10.1006/jmbi.1998.2402
DO - 10.1006/jmbi.1998.2402
M3 - Article
C2 - 9917410
AN - SCOPUS:0033613814
SN - 0022-2836
VL - 285
SP - 1765
EP - 1776
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 4
ER -