Role of proline isomerization in folding of ribonuclease A at low temperatures

Abstract

In unfolded RNase A there is an interconversion between slow-folding and fast-folding forms (Us UF) that is known to show properties characteristic of proline isomerization in model peptides. Here, we accept the evidence that Us molecules contain nonnative proline isomers and we ask about the isomerization of these proline residues during folding. The Us UF reaction in unfolded RNase A is used both to provide data on the kinetics of proline isomerization in the unfolded protein and as the basis of an assay for measuring proline isomerization during folding.The tyrosine-detected folding kinetics at low temperatures have been compared to those of proline isomerization in unfolded RNase A. The comparison is based on the recent observation that the Us UF kinetics are independent of guanidinium chloride concentration, so that they can be extrapolated to low guanidinium chloride concentrations, at which folding takes place. At 00C the tyrosine-detected folding reaction is 100-fold faster than the conversion of Us to UF in unfolded RNase A. Consequently, the folding reaction is not rate-limited by proline isomerization as it occurs in unfolded RNase A.An assay is given for proline isomerization during folding. The principle is that native RNase A yields UF on unfolding, whereas protein molecules that still contain nonnative proline isomers yield Us. Unfolding takes place at 0C, at which proline isomerization is slow compared to unfolding. This assay yields two important results: (i) The kinetics of proline isomerization during folding are substantially faster than in unfolded RNase A-e.g., 40-fold at 0C. The mechanism of the rate enhancement is unknown. (ii) At low temperatures (0-100C), and also in the presence of (NH42S04, the tyrosine-detected folding reaction occurs before proline isomerization and yields a folded intermediate IN that is able to bind the specific inhibitor 2S-CMP. The results demonstrate that a folding intermediate is spectrally detectable when folding occurs at low temperatures. They suggest that low temperatures provide suitable conditions for determining the kinetic pathway of folding by characterizing folding intermediates.In unfolded RNase A there is an equilibrium between fastfolding (UF) and slow-folding (Us) forms (1-6). The kinetics of the UF Us reaction can be measured by "double-jump" experiments (4, 7). First, native RNase A (N) is unfolded in a reaction that yields UF. Under suitable conditionst this is the unfolding reaction measured by the change in tyrosine absorbance (7). When unfolding occurs at 00C, an almost quantitative conversion of N to UF can be obtained before there is a significant conversion of UF to Us, as we show here. Afterwards, the kinetics of the slow equilibration of UF

The effect of methanol and temperature on the kinetics of refolding of ribonuclease A

“…Again, the rates were identical within experimental error. The rates of the slow refolding phases under our conditions of 0% methanol are in good agreement with such rates previously reported under reasonably similar conditions [8,15,18].…”

Cis proline mutants of ribonuclease A. II. Elimination of the slow‐folding forms by mutation

“…In future work, it will be important to use other spectroscopic probes, in addition to CD, to monitor the refolding of the double mutant and of the Pro 114 single mutants; it is particularly important to determine accurately the (UF):(Us) ratio in the Pro 114 single mutants. The UsII species is known to fold rapidly in strongly native conditions and to form the native-like intermediate IN before proline isomerization occurs (Cook et al, 1979;Schmid & Blaschek, 1981;Schmid, 1983). Moreover, the fluorescence of Tyr 92 is known to monitor the UF o Us reaction in unfolded RNase A (Rehage & Schmid, 1982;Schmid et al, 1986).…”

“…Proof that the folded species IN is formed before isomerization occurs is provided by a direct assay based on first unfolding, then refolding, the sample: IN unfolds to give Us, whereas N unfolds to give UF (Cook et al, 1979). Tyrosine fluorescence can be used to monitor the UF H Us reaction in unfolded RNase A (Rehage & Schmid, 1982) and Tyr 92, which is adjacent to the cis proline residue P93 (Kinemage 3), contributes strongly to the observed signal (Schmid et al, 1986).…”

“…The intermediate IN is native-like in most physical properties, including the specific binding of the substrate analog 2'CMP (Cook et al, 1979;Schmid & Blaschek, 1981) and IN has RNase catalytic activity (Schmid & Blaschek, 1981). Proof that the folded species IN is formed before isomerization occurs is provided by a direct assay based on first unfolding, then refolding, the sample: IN unfolds to give Us, whereas N unfolds to give UF (Cook et al, 1979).…”

“…The model proposes that the slow-folding property of a Us species is explained by slow proline isomerization (Us --t U,) at the first step of Us refolding. The major Us species of RNase A (UsII) was found, however, to fold in strongly native conditions by a pathway in which folding precedes isomerization (Cook et al, 1979;Schmid & Blaschek, 1981):…”