FOLDING PATHWAY OF ESCHERICHIA-COLI RIBONUCLEASE HI - A CIRCULAR-DICHROISM, FLUORESCENCE, AND NMR-STUDY

The unfolding and refolding processes of Escherichia coli ribonuclease HI at 25 degrees C, induced by concentration jumps of either guanidin e hydrochloride (GuHCl) or urea, were investigated using stopped-flow circular dichroism (CD), stopped-flow fluorescence, and NMR spectrosco pies. Only a single exponential process was detected for the fast time scale unfolding (rate constants from 0.014 to 0.54 s(-1) depending on the final denaturant concentration). For refolding, the far-UV CD val ue largely recovered within 50 ms of the stopped-flow mixing dead time (burst phase). This phase was followed by either one or two phases, w ith rate constants from 0.035 to 2.45 s(-1) as detected by CD and fluo rescence, respectively. Although this protein has a single cis-Pro res idue, a very slow phase due to proline isomerization was not observed, for either unfolding or refolding. The difference in the amplitudes o f the burst phases for refolding in the far- and near-UV CD spectra re vealed that an intermediate state exists, with the characteristics of a molten globule. Because the one-phased fast exponential process dete cted by CD corresponds to the slower of the two phases detected by flu orescence, the intermediate detected by CD might be the most stable. G uHCl denaturation experiments revealed that this intermediate cooperat ively unfolds, with a transition midpoint of 1.33 +/- 0.03 M. The Gibb s free energy difference (Delta G) between the intermediate and the un folded states, under physiological conditions (25 degrees C, pH 5.5, a nd 0 M GuHCl), was estimated to be 20.0 +/- 2.3 kJ mol(-1) Therefore, it is reasonable to assume that the refolding intermediate, rather tha n the unfolded state, is the latent denatured state under physiologica l conditions. Approximately linear relationships between the GuHCl con centration and the logarithm of the microscopic rate constants determi ned by CD and fluorescence were also observed. By extrapolation to a G uHCl concentration of 0 M, activation Gibbs free energies of 98.5 +/- 1.1 kJ mol(-1) for unfolding and 69.5 +/- 0.2 kJ mol(-1) for refolding under physiological conditions were obtained. The hydrogen-exchange-r efolding competition combined with two-dimensional NMR revealed that t he amide protons of a-helix I are the most highly protected, suggestin g that alpha-helix I is the initial site of protein folding. The CD an d NMR data showed that the intermediate state has a structure similar to that of the acid-denatured molten globule.