The chaperonin GroEL binds to late-folding non-native conformations present in native Escherichia coli and murine dihydrofolate reductases

Dihydrofolate reductases from mouse (MuDHFR) or Escherichia coli (EcDHFR) a re shown to refold via several intermediate forms, each of which can bind t o the chaperonin GroEL. When stable complexes with GroEL are formed, they c onsist of late-folding intermediates. In addition, we find that late-foldin g intermediates that are present in the native enzyme bind to GroEL. For th e E, coli and murine proteins, the extent of protein bound increases as the temperature is increased from 8 degrees C to 42 degrees C, at which temper ature either protein is completely bound as the last (EcDHFR) or the last t wo (MuDHFR)folding intermediate(s). Thus for EcDHFR, the binding is transie nt at low temperature (<30 degrees C) and stable at high temperature (>35 d egrees 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 fo r the first step in DHFR unfolding, which is unfavorable, and the free ener gy of binding the nonnative conformation, which is favorable. For EcDHFR, t his results in an overall binding free energy that is unfavorable below 30 degrees C. Over the temperature range of 8 degrees C to 42 degrees C, GroEL binds MuDHFR more tightly than EcDHFR, due partially to a small free energ y difference between two pre-existing non-native conformations of MuDHFR, r esulting in binding to more than one folding intermediate. (C) 1999 Academi c Press.