The role of proline residues in the folding kinetics of the bovine pancreatic trypsin inhibitor derivative RCAM(14-38).
The role of proline residues in the folding kinetics of the bovine pancreatic trypsin inhibitor derivative RCAM(14-38).
复制标题
脯氨酸残基在牛胰蛋白酶抑制剂衍生物 RCAM(14-38) 折叠动力学中的作用。
DOI：
10.1016/0022-2836(81)90343-0
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发表时间：
1981
影响因子：
5.6
通讯作者：
Baldwin,RL
中科院分区：
文献类型：
--
作者：
Jullien,M;Baldwin,RL
通过对未折叠蛋白质的慢折叠物种的测试，研究了Pro残基在胰酶抑制剂衍生物RCAM(14-38)折叠中的作用，这可能是由于在展开后引入了错误的Pro异构体造成的。在25°C下未折叠的蛋白质主要含有快速折叠(UF)分子：在1.9M-氯化胍中，在pH 6.8时，它们以40毫秒的时间常数进行折叠。至少已经发现了一个较小的缓慢折叠(US)物种，使用荧光来监测折叠。在展开后形成这个用户物种的反应显示了预期的性质：脯氨酸残基的伊朗聚合。当用酪氨酸吸光度监测复性时，发现了两个轻微的缓慢反应。较快的反应发生在与荧光法相同的时间范围内(25℃下15 S)，而较慢的反应较慢(25℃下200 S)。目前尚不清楚这种较慢的反应是不是由第二个Us种引起的。牛胰蛋白酶抑制物中有四种反式脯氨酸残基：如果每一个脯氨酸残基都能产生一个Us物种，则慢折叠分子(在25℃时不超过25%)的比例比预期的要小，如果每个残基的去折叠后的转化率至少为0.1：0.9，则根据折叠动力学给出了含有一个错误的脯氨酸异构体的未折叠分子所表现出的折叠反应类型的分类标准。Levitt(1980)根据天然蛋白质和含有错误的Pro异构体的预测的最小能量结构之间的能量差(小的、中的或大的)将Pro残基分为三种类型。他认为，这三种类型的脯氨酸残基可以通过它们产生的折叠反应的类型来识别。到目前为止，只有第二类(中间)折叠反应被用这里介绍的标准来描述。我们指出，折叠反应的类型还取决于折叠条件，并对这种效应给出了可能的解释。
The role of proline residues in the folding of the trypsin inhibitor derivative RCAM(14–38) has been studied by testing for slow-folding species of the unfolded protein, which could result from the introduction of wrong proline isomers after unfolding. The unfolded protein at 25 °C contains chiefly fast-folding (UF) molecules: they refold with a time constant of 40 milliseconds at pH 6.8 in 1.9m-guanidinium chloride. At least one minor slow-folding (Us) species has been found, using fluorescence to monitor refolding. The reaction in which this Usspecies is formed after unfolding shows the properties expected for thecis: Iransisomerization of a proline residue. When refolding is monitored by tyrosine absorbance, two minor slow reactions are found. The faster reaction is in the same time range (15 s at 25 °C) as that studied by fluorescence, and the slower reaction is quite slow (200 s at 25 °C). It is not known whether the slower reaction results from a second Usspecies. There are fourtransproline residues in bovine pancreatic trypsin inhibitor: the proportion of slow-folding molecules (not more than 25% at 25 °C) is smaller than expected if every proline residue can produce a Usspecies and if thecistotransratio of each residue after unfolding is at least 0.1:0.9.Criteria based on folding kinetics are given for classifying the types of folding reaction shown by unfolded molecules containing a single wrong proline isomer. Levitt (1980) has classified three types of proline residues according to the energy difference (small, intermediate or large) between the native protein and the predicted minimum energy structure containing a wrong proline isomer. He suggests that these three types of proline residues can be recognized by the types of folding reactions they produce. Only type II (intermediate) folding reactions have thus far been characterized by the criteria introduced here. We point out that the type of folding reaction depends also on the folding conditions, and a possible explanation for this effect is given.