Observation of intermediates in the folding of ribonuclease A at low temperature using proton nuclear magnetic resonance.
Observation of intermediates in the folding of ribonuclease A at low temperature using proton nuclear magnetic resonance.
复制标题
使用质子核磁共振观察核糖核酸酶 A 低温折叠过程中的中间体。
DOI：
10.1021/bi00262a035
复制
发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
Fink,AL
中科院分区：
文献类型：
--
作者：
Biringer,RG;Fink,AL
摘要：研究了核糖核酸酶A(RNaseA)在0~-20℃的甲醇水溶液中的复性。当将热展开的样品在复性条件下(例如，-16℃，35%甲醇，pH*2.8)进行复性时，根据286 nm处的吸光度变化(反映Tyr的溶剂暴露程度)，复性是三相的，大约需要1h完成。天然酶在35%或50%甲醇水溶液中的360 MHz核磁共振谱与在水溶液中的核磁共振谱非常相似。当用信号在零下温度范围内监测RNaseA的折叠时，有证据表明，RNaseA1等小蛋白的折叠是中间控制的(Cook等人，1979；Schmid，1981；Schmidd&Baldwin，1979；Blum等人，1978)；也就是说，它是由部分折叠的中间态的结构和稳定性决定的(Ptitsyn&Rashin，1975)。本研究旨在阐明这类中间物种的性质。核糖核酸酶A的复性已经被广泛地研究，特别是Baldwin和他的同事[如Baldwin(1975,1980)和Baldwin&Creighton(1980)]，在各种条件下在水溶液中。据报道，至少有两种不同的中间体存在。根据酰胺质子交换和折叠的竞争实验，Schmidd&Baldwin(1979)证明了存在早期的中间结构，缺乏显著的致密、球状结构。库克等人(1979)和施密德(1981；Schmidd&Blaschek，1981)进行的一系列广泛的实验表明，在“强烈”的自然条件下折叠时，会形成一种类似于本地的中间体IN。Pro-93被认为是IN中的非天然反式构型。这种中间体可以结合特定的抑制剂，具有催化活性，并被认为具有与天然蛋白几乎相同的结构，但在Pro-93周围的环附近除外。
Roger G. Biringer and Anthony L. Fink* abstract: The refolding of ribonuclease A (RNase A) has been investigated in aqueous methanol cryosolvents in the 0 to-20 C range. When a thermally unfolded sample was brought under renaturing conditions (eg,-16 C, 35% methanol, pH* 2.8), the refolding, as monitored by the absorbance change at 286 nm (which reflects thedegree of solvent exposure of Tyr), was triphasic and took approximately 1 h for completion. The 360-MHz protonnuclear magnetic resonance(NMR) spectrum of the native enzyme in either 35% or 50% aqueous methanol is very similar to that in aqueous solution. When therefolding of RNase A was monitored in the subzero temperature range with the signals liecent evidence suggests that the folding of small proteins such as RNase A1 is intermediate controlled (Cook et al., 1979; Schmid, 1981; Schmid & Baldwin, 1979; Blum et al., 1978); that is, it is determined by the structure and stability of partially folded intermediate states (Ptitsyn & Rashin, 1975). The present study was undertaken to shed light on the nature of such intermediate species. The refolding of RNase A has been extensively studied, especially by Baldwin and co-workers [eg, Baldwin (1975, 1980) and Baldwin & Creighton (1980)], under a variety of conditions in aqueous solutions. Evidence for at least two different intermediates has been reported. On the basis of competition experiments between amide proton exchange and folding, Schmid & Baldwin (1979) demonstrated the presence of an early intermediate, lacking significant compact, globular structure. An extensive series of experiments by Cook et al.(1979) and Schmid (1981; Schmid & Blaschek, 1981) has shown that a nativelike intermediate, IN, is formed during folding under “strongly” native conditions. Pro-93 has been proposed to be in the nonnative trans configuration in IN. This intermediate can bind specific inhibitors, is catalytically active, and is presumed to have a structure virtually identical with that of the native protein except in the immediate vicinity of the loop around Pro-93.