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 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


Submitter: Connie Wang

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

Version: 1

Publication Details
Yamasaki K;Ogasahara K;Yutani K;Oobatake M;Kanaya S,Biochemistry (1995) Folding pathway of Escherichia coli ribonuclease HI: a circular dichroism, fluorescence, and NMR study. PMID:8527428
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Ribonuclease HI A7ZHV1 RNH_ECO24
100.0 Ribonuclease HI B7MBJ0 RNH_ECO45
100.0 Ribonuclease HI P0A7Y6 RNH_ECO57
100.0 Ribonuclease HI B5Z0I8 RNH_ECO5E
100.0 Ribonuclease HI B7NKW4 RNH_ECO7I
100.0 Ribonuclease HI B7MQ23 RNH_ECO81
100.0 Ribonuclease HI B7M213 RNH_ECO8A
100.0 Ribonuclease HI C4ZRV1 RNH_ECOBW
100.0 Ribonuclease HI B1XD78 RNH_ECODH
100.0 Ribonuclease HI P0A7Y5 RNH_ECOL6
100.0 Ribonuclease HI B1IPU4 RNH_ECOLC
100.0 Ribonuclease HI P0A7Y4 RNH_ECOLI
100.0 Ribonuclease HI B7N876 RNH_ECOLU
100.0 Ribonuclease HI B6HZS7 RNH_ECOSE
100.0 Ribonuclease HI B1LHM3 RNH_ECOSM
100.0 Ribonuclease HI B7LW89 RNH_ESCF3
100.0 Ribonuclease HI B2U352 RNH_SHIB3
100.0 Ribonuclease HI Q325T2 RNH_SHIBS
100.0 Ribonuclease HI Q32JP9 RNH_SHIDS
100.0 Ribonuclease HI P0A7Y7 RNH_SHIFL
100.0 Ribonuclease HI Q3Z5E9 RNH_SHISS
99.4 Ribonuclease HI B7UJB0 RNH_ECO27
99.4 Ribonuclease HI B7LHC0 RNH_ECO55
99.4 Ribonuclease HI A7ZWF6 RNH_ECOHS
99.4 Ribonuclease HI Q0TLC3 RNH_ECOL5
93.5 Ribonuclease HI A8AKR0 RNH_CITK8
93.5 Ribonuclease HI B5F8X2 RNH_SALA4
93.5 Ribonuclease HI A9MPF1 RNH_SALAR
93.5 Ribonuclease HI Q57SZ6 RNH_SALCH
93.5 Ribonuclease HI B5FJ58 RNH_SALDC
93.5 Ribonuclease HI B5R449 RNH_SALEP
93.5 Ribonuclease HI B5R5L3 RNH_SALG2
93.5 Ribonuclease HI B4TK85 RNH_SALHS
93.5 Ribonuclease HI B4SV39 RNH_SALNS
93.5 Ribonuclease HI Q5PFD8 RNH_SALPA
93.5 Ribonuclease HI A9MZ19 RNH_SALPB
93.5 Ribonuclease HI B5BDW5 RNH_SALPK
93.5 Ribonuclease HI B4TYH0 RNH_SALSV
93.5 Ribonuclease HI P0A2C0 RNH_SALTI
93.5 Ribonuclease HI P0A2B9 RNH_SALTY
92.9 Ribonuclease HI C0Q6N2 RNH_SALPC
90.9 Ribonuclease HI B5Y1G2 RNH_KLEP3
90.9 Ribonuclease HI A6T512 RNH_KLEP7