Secondary structure in ribonuclease: I. Equilibrium folding transitions seen by amide circular dichroism

Abstract

The amide circular dichroism (c.d.) spectra of RNAase A, RNAase S and S-protein were measured and analyzed in the wavelength region from 200 to 260 nm. The range is sufficient to quantitate reliably the apparent fractional contents of α-helix and antiparallel β-sheet. Extraction of additional structural information was not attempted (e.g. the apparent fraction of β-turns). Various decomposition procedures were tested and gave the same results. Three spectroscopically well-distinct denaturation transitions were detected and characterized: (1) acid denaturation; (2) thermal denaturation; (3) denaturant induced unfolding. 1. (1) Lowering the pH from 6.8 to 1.7 (at 10 °C) results in the loss of some 9% helical content for RNAase S; no change in structural composition was detected for RNAase A. The 9% loss in helix is consistent with the disruption of the S-peptide α-helix of residues 3 to 12. It is known that S-peptide dissociates at low pH values (Richards & Logue, 1962). 2. (2) Thermal denaturation at pH 6.8 or 1.7 destroys the β-structure in RNAase A, RNAase S or S-protein. A fractional change of 36 to 38% is detected, which agrees favorably with the entire β-sheet content (40%) (Wyckoff et al., 1970). In addition, thermal denaturation of RNAase A at pH 6.8 or 1.7 results in a fractional loss of 9% α-helix. No change in apparent helical content is detected for S-protein in the same conditions, although the folded S-protein moiety accounts for almost two-thirds of the helical content of ribonuclease. RNAase S behaves during thermal denaturation: at low pH the same as S-protein; at neutral pH like RNAase A, with a lower, concentration-dependent melting temperature ( t m), however. 3. (3) Random coil c.d. spectra are generated by subjecting thermally denatured or reduced and carboxymethylated RNAase A, RNAase S or S-protein to proteolytic digestion by trypsin (at neutral pH) or pepsin (at pH 1.7). A c.d. spectrum very similar to that of digested material is obtained with undigested material in 9 m-urea or 6 m-guanidine hydrochloride (GuHCl). The difference between the spectra of thermally denatured material and material denatured by 6 m-GuHCl is consistent in shape with an α-helix to coil difference spectrum, and in magnitude with a fraction of 10 to 12% of the residues being involved. The latter figure and the 9% change in helical content assigned to the denaturation of the S-peptide add up satisfactorily to the 21% helical content of RNAase S in the crystal structure (Wyckoff et al., 1970). Comparing the changes in apparent structural composition during denaturation under various conditions obtained for RNAase A, RNAase S and S-protein allows one to analyze the mutual stabilization of the different secondary structural elements. (1) The folded β-sheet is necessary in order to observe folding of the S-peptide helix. It has been shown earlier that (low affinity) fragment association is not sufficient (Labhardt, 1981). (2) Folding of the β-sheet alone cannot stabilize the entire helical content of the S-protein moiety in the presence of GuHCl. (3) The folded helical S-peptide stabilizes both, the β-sheet and the entire S-protein moiety helix content.