Cook K H, Schmid F X, Baldwin R L
Proc Natl Acad Sci U S A. 1979 Dec;76(12):6157-61. doi: 10.1073/pnas.76.12.6157.
In unfolded RNase A there is an interconversion between slow-folding and fast-folding forms (U(S) right harpoon over left harpoon U(F)) that is known to show properties characteristic of proline isomerization in model peptides. Here, we accept the evidence that U(S) molecules contain nonnative proline isomers and we ask about the isomerization of these proline residues during folding. The U(S) right harpoon over left harpoon U(F) 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 U(S) right harpoon over left harpoon U(F) kinetics are independent of guanidinium chloride concentration, so that they can be extrapolated to low guanidinium chloride concentrations, at which folding takes place. At 0 degrees C the tyrosine-detected folding reaction is 100-fold faster than the conversion of U(S) to U(F) 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 U(F) on unfolding, whereas protein molecules that still contain nonnative proline isomers yield U(S). Unfolding takes place at 0 degrees C, 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 0 degrees C. The mechanism of the rate enhancement is unknown. (ii) At low temperatures (0-10 degrees C), and also in the presence of (NH(4))(2)SO(4), the tyrosine-detected folding reaction occurs before proline isomerization and yields a folded intermediate I(N) that is able to bind the specific inhibitor 2'-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.
在去折叠的核糖核酸酶A中，慢折叠形式和快折叠形式之间存在相互转换（U(S) ⇌ U(F)），已知这种转换表现出模型肽中脯氨酸异构化的特征性质。在此，我们认可U(S)分子含有非天然脯氨酸异构体这一证据，并探讨这些脯氨酸残基在折叠过程中的异构化情况。去折叠的核糖核酸酶A中的U(S) ⇌ U(F)反应既用于提供去折叠蛋白质中脯氨酸异构化动力学的数据，也作为测量折叠过程中脯氨酸异构化的一种检测方法的基础。已将低温下酪氨酸检测到的折叠动力学与去折叠的核糖核酸酶A中脯氨酸异构化的动力学进行了比较。这种比较基于最近的观察结果，即U(S) ⇌ U(F)动力学与氯化胍浓度无关，因此可以外推到低氯化胍浓度，此时折叠发生。在0℃时，酪氨酸检测到的折叠反应比去折叠的核糖核酸酶A中U(S)向U(F)的转换快100倍。因此，折叠反应不像在去折叠的核糖核酸酶A中那样受脯氨酸异构化的速率限制。给出了一种用于测量折叠过程中脯氨酸异构化的检测方法。原理是天然核糖核酸酶A在去折叠时产生U(F)，而仍含有非天然脯氨酸异构体的蛋白质分子产生U(S)。去折叠在0℃进行，此时与去折叠相比脯氨酸异构化较慢。这种检测方法产生了两个重要结果：（i）折叠过程中脯氨酸异构化的动力学比去折叠的核糖核酸酶A中的快得多，例如在0℃时快40倍。速率增强的机制尚不清楚。（ii）在低温（0 - 10℃）下，以及在(NH₄)₂SO₄存在的情况下，酪氨酸检测到的折叠反应在脯氨酸异构化之前发生，并产生一种能够结合特异性抑制剂2'-CMP的折叠中间体I(N)。结果表明，当在低温下发生折叠时，折叠中间体在光谱上是可检测的。它们表明低温为通过表征折叠中间体来确定折叠的动力学途径提供了合适的条件。
