Alexander M. Labhardt

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

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 (tm), 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.

Recombination of S-peptide with S-protein during folding of ribonuclease S: I. Folding pathways of the slow-folding and fast-folding classes of unfolded S-protein☆

Abstract The refolding kinetics of ribonuclease S have been measured by tyrosine absorbance, by tyrosine fluorescence emission, and by rapid binding of the specific inhibitor 2′CMP † to folded RNAase S. The S-protein is first unfolded at pH 1.7 and then either mixed with S-peptide as refolding is initiated by a stopped-flow pH jump to pH 6.8, or the same results are obtained if S-protein and S-peptide are present together before refolding is initiated. The refolding kinetics of RNAase S have been measured as a function of temperature (10 to 40 °C) and of protein concentration (10 to 120 μ m ). The results are compared to the folding kinetics of S-protein alone and to earlier studies of RNAase A. A thermal folding transition of S-protein has been found below 30 °C at pH 1.7; its effects on the refolding kinetics are described in the following paper (Labhardt & Baldwin, 1979). In this paper we characterize the refolding kinetics of unfolded S-protein, as it is found above 30 °C at pH 1.7, together with the kinetics of combination between S-peptide and S-protein during folding at pH 6.8. Two classes of unfolded S-protein molecules are found, fast-folding and slow-folding molecules, in a 20: 80 ratio. This is the same result as that found earlier for RNAase A; it is expected if the slow-folding molecules are produced by the slow cis-trans isomerization of proline residues after unfolding, since S-protein contains all four proline residues of RNAase A. The refolding kinetics of the fast-folding molecules show clearly that combination between S-peptide and S-protein occurs before folding of S-protein is complete. If combination occurred only after complete folding, then the kinetics of formation of RNAase S should be rather slow (5 s and 100 s at 30 °C) and nearly independent of protein concentration, as shown by separate measurements of the folding kinetics of S-protein, and of the combination between S-peptide and folded S-protein. The observed folding kinetics are faster than predicted by this model and also the folding rate increases strongly with protein concentration (apparent 1.6 order kinetics). The fact that RNAase S is formed more rapidly than S-protein alone is sufficient by itself to show that combination with S-peptide precedes complete folding of S-protein. Computer simulation of a simple, parallel-pathway scheme is able to reproduce the folding kinetics of the fast-folding molecules. All three probes give the same folding kinetics. These results exclude the model for protein folding in which the rate-limiting step is an initial diffusion of the polypeptide chain into a restricted range of three-dimensional configurations (“nueleation”) followed by rapid folding (“propagation”). If this model were valid, one would expect comparable rates of folding for RNAase A and for S-protein and one would also expect to find no populated folding intermediates, so that combination between S-peptide and S-protein should occur after folding is complete. Instead, RNAase A folds 60 times more rapidly than S-protein and also combination with S-peptide occurs before folding of S-protein is complete. The results demonstrate that the folding rate of S-protein increases after the formation, or stabilization, of an intermediate which results from combination with S-peptide. They support a sequential model for protein folding in which the rates of successive steps in folding depend on the stabilities of preceding intermediates. The refolding kinetics of the slow-folding molecules are complex. Two results demonstrate the presence of folding intermediates: (1) the three probes show different kinetic progress curves, and (2) the folding kinetics are concentration-dependent, in contrast to the results expected if complete folding of S-protein precedes combination with S-peptide. A faster phase of the slow-refolding reaction is detected both by tyrosine absorbance and fluorescence emission but not by 2′CMP binding, indicating that native RNAase S is not formed in this phase. Comparison of the kinetic progress curves measured by different probes is made with the use of the kinetic ratio test , which is defined here.

Recombination of S-peptide with S-protein during folding of ribonuclease S. II. Kinetic characterization of a stable folding intermediate shown by S-protein at pH 1.7.

Abstract At pH 1.7 S-peptide dissociates from S-protein but S-protein remains partly folded below 30 °C. A folded form of S-protein, labeled I 3 , is detected and measured by its ability to combine rapidly with S-peptide at pH 6.8 and then to form native ribonuclease S. The second-order combination reaction ( k = 0.7 × 10 6 m −1 s −1 at 20 °C) can be monitored either by tyrosine absorbance or fluorescence emission; the subsequent first-order folding reaction (half-time, 68 ms; 20 °C) is monitored by 2′CMP † binding. Combination with S-peptide and folding to form native RNAase S is considerably slower for both classes of unfolded S-protein (see preceding paper). I 3 shows a thermal folding transition at pH 1.7: it is completely unfolded above 32 °C and reaches a limiting low-temperature value of 65% below 10 °C. The 35% S-protein remaining at 10 °C is unfolded as judged by its refolding behavior in forming native RNAase S at pH 6.8. The folding transition of S-protein at pH 1.7 is a broad, multi-state transition. This is shown both by the large fraction of unfolded S-protein remaining at low temperatures and by the large differences between the folding transition curves monitored by I 3 and by tyrosine absorbance. The fact that S-protein remains partly folded after dissociation of S-peptide at pH 1.7 but not at pH 6.8 may be explained by two earlier observations. (1) Native RNAase A is stable in the temperature range of the S-protein folding transition at pH 1.7, and (2) the binding constant of S-protein for S-peptide falls steadily as the pH is lowered, by more than four orders of magnitude between pH 8.3 and pH 2.7, at 0 °C. The following explanation is suggested for why folding intermediates are observed easily in the transition of S-protein but not of RNAase A. The S-protein transition is shifted to lower temperatures, where folding intermediates should be more stable: consequently, intermediates in the folding of RNAase A which do not involve the S-peptide moiety and which are populated to almost detectable levels can be observed at the lower temperatures of the S-protein transition.

Design and synthesis of novel nonpolar host peptides for the determination of the 310- and α-helix compatibilities of α-amino acid buildig blocks: An assessment of α,α-disubstituted glycines

The present work describes three novel nonpolar host peptide sequences that provide a ready assessment of the 310‐ and α‐helix compatibilities of natural and unnatural amino acids at different positions of small‐ to medium‐size peptides. The unpolar peptides containing Ala, Aib, and a C‐terminal p‐iodoanilide group were designed in such a way that the peptides could be rapidly assembled in a modular fashion, were highly soluble in solvent mixtures of triflouroethanol and H2O for CD‐ and two‐dimensional (2D) nmr spectroscopic analyses, and showed excellent crystallinity suited for x‐ray structure analysis. To validate our approach we synthesized 9‐mer peptides 79a–96 (Table IV), 12‐mer peptides 99–110c (Table V), and 10‐mer peptides 120a–125d and 129–133 (Table VI and Scheme 8) incorporating a series of optically pure cyclic and open‐chain (R)‐ and (S)‐α,α‐disubstituted glycines 1–10 (Figure 2). These amino acids are known to significantly modulate the conformations of small peptides.

Secondary structure in ribonuclease: II. Relations between folding kinetics and secondary structure elements

The kinetics of regain of 2′-CMP binding are monitored during renaturation of RNAase S. Experiments were performed by mixing equimolar amounts of S-peptide with S-protein. The S-protein fragment was incubated initially (i.e. before mixing with S-peptide) at pH 6.2 or 1.7 and various guanidine hydrochloride (GuHCl) concentrations. Three well-resolved phases are observed. The fastest phase is second-order. The reciprocal half-time increases linearly with fragment concentration and is independent of initial conditions for the S-protein fragment. An apparent on rate of kon = 2 × 105m−1s−1 is measured in 0.5 m-GuHCl (pH 6.2) and 20 ° C. Identical association kinetics are observed by changes in tyrosine absorbance. The fraction of native RNAase S formed in this second-order reaction strictly equals the fraction of S-protein molecules with intact β-sheet in initial conditions. The relation holds for different pH values, GuHCl concentrations and temperatures. The fraction of apparent helical content of S-protein in initial conditions may also vary but this is not reflected by the association reaction. We interpret this to mean that the β-sheet but not the α-helices must be preformed in initial conditions in order to generate the high-affinity peptide binding site of S-protein. Furthermore, it is concluded that the S-protein moiety β-sheet forms or unfolds in a single one-step reaction. 2′-CMP binding reports, additionally, two slower phases of renaturation. These are produced by S-protein molecules that have their β-sheet unfolded in initial conditions. It is observed that a unique dependence of these two folding rates exists for RNAase A, RNAase S and S-protein as function of tm, the temperature of half-completion of thermal denaturation as measured by unfolding of the β-sheet in the respective compound in final conditions. The tm value varies with changing pH, with GuHCl concentration and (for RNAase S) with changing fragment concentration. The findings are interpreted to argue in favor of a sequential mechanism of folding, where the stability of a structural precursor determines the rate of folding.