Abstract

The unfolding and refolding processes of Escherichia coli ribonuclease HI at 25 degrees C, induced by concentration jumps of either guanidine hydrochloride (GuHCl) or urea, were investigated using stopped-flow circular dichroism (CD), stopped-flow fluorescence, and NMR spectroscopies. 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 value largely recovered within 50 ms of the stopped-flow mixing dead time (burst phase). This phase was followed by either one or two phases, with rate constants from 0.035 to 2.45 s-1 as detected by CD and fluorescence, respectively. Although this protein has a single cis-Pro residue, a very slow phase due to proline isomerization was not observed, for either unfolding or refolding. The difference in the amplitudes of the burst phases for refolding in the far- and near-UV CD spectra revealed that an intermediate state exists, with the characteristics of a molten globule. Because the one-phased fast exponential process detected by CD corresponds to the slower of the two phases detected by fluorescence, the intermediate detected by CD might be the most stable. GuHCl denaturation experiments revealed that this intermediate cooperatively unfolds, with a transition midpoint of 1.33 +/- 0.03 M. The Gibbs free energy difference (delta G) between the intermediate and the unfolded states, under physiological conditions (25 degrees C, pH 5.5, and 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 than the unfolded state, is the latent denatured state under physiological conditions. Approximately linear relationships between the GuHCl concentration and the logarithm of the microscopic rate constants determined by CD and fluorescence were also observed. By extrapolation to a GuHCl 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-refolding competition combined with two-dimensional NMR revealed that the amide protons of alpha-helix I are the most highly protected, suggesting that alpha-helix I is the initial site of protein folding. The CD and NMR data showed that the intermediate state has a structure similar to that of the acid-denatured molten globule. Study holds ProTherm entries: 4927 Extra Details: cis-Pro residue; proline isomerization; intermediate state;,cooperative; hydrogen-exchange-refolding; molten globule

Submission Details

ID: voL4KZRD3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:27 p.m.

Version: 1

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