The unfolding of ribonuclease A was studied in 5.2 M guanidine hydrochloride at pH 8 and 10 degrees C using multiple optical probes, native-state hydrogen exchange (HX), and pulse labeling by hydrogen exchange. First, native-state HX studies were used to demonstrate that the protein exists in two slowly interconverting forms under equilibrium native conditions: a predominant exchange-incompetent N form and an alternative ensemble of conformations, N(I), in which some amide hydrogens are fully exposed to exchange. Pulsed HX studies indicated that, during unfolding, the rates of exposure to exchange with solvent protons were similar for all backbone NH probe protons. It is shown that two parallel routes of unfolding are available to the predominant N conformation as soon as it encounters strong unfolding conditions. A fraction of molecules appears to rapidly form N(I) on one route. On the other route an exchange-incompetent intermediate state ensemble, I(U)(2), is formed. The kinetics of unfolding measured by far-UV circular dichroism (CD) were faster than those measured by near-UV CD and intrinsic tyrosine fluorescence of the protein. The logarithms of the rate constants of the unfolding reaction measured by all three optical probes also showed a nonlinear dependence on GdnHCl concentration. All of the data suggest that N(I) and I(U)(2) are nativelike in their secondary and tertiary structures. While N(I) unfolds directly to the fully exchange-competent unfolded state (U), I(U)(2) forms another intermediate I(U)(3) which then unfolds to U. I(U)(3) is devoid of all native alpha-helical secondary structure and has only 30% of the tertiary interactions still intact. Since the rates of global unfolding measured by near-UV CD and fluorescence agree well with the rates of exposure determined for all of the backbone NH probe protons, it appears that the rate-limiting step for the unfolding of RNase A is the dissolution of the entire native tertiary structure and penetration of water into the hydrophobic core.
Barstar is known to form a molten globule-like A form below pH 4. This form exists as a soluble aggregate of 16 monomeric subunits, and appears to remain homogeneous in solution for at least two weeks. Here, structural characterization by NMR of the flexible regions in the A form of barstar has been carried out at pH 2.7 and 25 degrees C. Significantly, the A form appears to be a symmetrical aggregate. Using the recently described fast assignment strategy from HNN and HN(C)N spectra, along with the standard triple resonance and three-dimensional NMR experiments, the flexible segment of the aggregate has been identified to belong largely to the N-terminal end of the polypeptide chain; sequential connectivities were obtained for the first 20 residues (except two) from these experiments. This segment is free in each of the monomeric subunits, and does not form a part of the aggregated core of the A form. The secondary chemical shifts of these residues suggest propensity toward an extended structure. Their (3)J(HN,H)(alpha) coupling constants have values corresponding to those in a random coil structure. However, a few medium-range NOEs, some of them involving side chain atoms, are observed between some residues in this segment. The lowered temperature coefficients of the H(N) chemical shifts compared to random coil values indicate possibilities of some hydrogen bonding in this region. Analysis of the (15)N relaxation parameters and reduced spectral density functions, in particular the negative values of heteronuclear NOEs, indicates large-amplitude high-frequency motions in the N-terminal segments; the first three residues show more negative NOEs than the others. The (15)N transverse relaxation rates and the J(0) spectral density values for residues Ser12 and Ser69 are significantly larger than for the rest, indicating some microsecond to millisecond time scale conformational exchange contributions to the relaxation of these residues. Taken all together, the data suggest that the A form of barstar is an aggregate with a rigid core, but with the N-terminal 20 residues of each of the monomeric subunits, in a highly dynamic random coil conformation which shows transient local ordering of structure. The N-terminal segment, anchored to the aggregated core, exhibits free-flight motion.
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