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首页> 外文期刊>Journal of Molecular Biology >Origin of apparent fast and non-exponential kinetics of lysozyme foldingmeasured in pulsed hydrogen exchange experiments
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Origin of apparent fast and non-exponential kinetics of lysozyme foldingmeasured in pulsed hydrogen exchange experiments

机译：脉冲氢交换实验中溶菌酶折叠的表观快速和非指数动力学的起源

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Folding of lysozyme at pH 5.2 is a complex processes. After rapid collapse (< 1 ms) kinetic partitioning into a slow and fast folding pathway occurs. The fast pathway leads directly to the native structure (N), whereas the slow pathway goes through a partially folded intermediate (I-1) with native-like secondary structure in the cc-domain. This mechanism is in agreement with data from a large number of spectroscopic probes, from changes in the radius of gyration and from measurements on the time-course of the populations of the different species. Results from pulsed hydrogen exchange experiments, in contrast, revealed that the secondary structure of I-1 and of N is formed significantly faster than changes in spectroscopic properties occur and showed large variations in the protection kinetics of individual amide sites. We investigated the molecular origin of the rapid amide protection by quantitatively simulating all kinetic processes during the pulse-labeling experiments. Absorbance and fluorescence-detected folding kinetics showed that the early events in lysozyme folding are accelerated under exchange conditions (pH 9.2) and that a change in folding mechanism occurs due to the transient population of an additional intermediate (I-1). This leads to kinetic competition between exchange and folding during the exchange pulse and to incomplete labeling of amide sites with slow intrinsic exchange rates. As a result, apparently faster and non-exponential kinetics of amide protection are measured in the labeling experiments. Our results further suggest that collapsed lysozyme (C) and I-1 have five and ten-times reduced free exchange rates, respectively, due to limited solvent accessibility.
机译：pH 5.2的溶菌酶折叠是一个复杂的过程。快速塌陷（<1 ms）后，发生动力学划分为慢速和快速折叠路径。快速途径直接导致天然结构（N），而缓慢途径通过部分折叠的中间体（I-1），其在cc-结构域具有天然样二级结构。这种机制与来自大量光谱探针的数据，回转半径的变化以及不同物种种群的时程测量结果相一致。相比之下，脉冲氢交换实验的结果表明，I-1和N的二级结构形成的速度明显快于光谱性质的变化，并且单个酰胺位点的保护动力学表现出较大的差异。我们通过定量模拟脉冲标记实验过程中的所有动力学过程，研究了快速酰胺保护的分子起源。吸光度和荧光检测的折叠动力学表明，在交换条件（pH 9.2）下，溶菌酶折叠的早期事件被加速，并且由于另外的中间体（I-1）的瞬时填充，折叠机理发生了变化。这导致交换脉冲期间交换和折叠之间的动力学竞争，并导致内酰胺交换速率慢的酰胺位点标记不完整。结果，在标记实验中测量到明显更快和非指数的酰胺保护动力学。我们的结果进一步表明，由于溶剂可及性有限，崩溃的溶菌酶（C）和I-1的自由交换率分别降低了五倍和十倍。

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