In pursuit of protein folding.
In pursuit of protein folding.
复制标题
追求蛋白质折叠。
DOI：
10.1126/science.8235606
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Englander,SW
中科院分区：
文献类型：
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作者：
Englander,SW
S.沃尔特·英格兰德，自然结构的形成。Miranker等人(1)用这些方法研究了鸡蛋溶菌酶的折叠。早期的氢交换（HX）标记实验表明，溶菌酶具有迄今为止观察到的大多数折叠行为的令人惊讶和特别令人沮丧的方面。折叠是异质的。在化学家的试管中，经过严格纯化的蛋白质制剂分裂成不同的亚组分，以不同的速度折叠。这种折叠的异质性导致了特殊的解释问题，米兰克等人通过巧妙地应用质谱法解决了这个问题。质谱法可以根据质量分离重折叠的蛋白质，因此与NMR分析不同，可以解析HX标记实验中获得的HD标记的分子分布。单链溶菌酶分子含有两个不同的叶：富含螺旋的a结构域和富含P折叠的P结构域。早期的工作（6）表明，对HX标记的保护作用在快速阶段（5至10 ms）发展，对于α结构域中40%的每种可测量的酰胺和P结构域中25%的每种可测量的酰胺。早期折叠阶段可能代表40%的分子仅折叠其a结构域，25%仅折叠其P结构域。在这种情况下，质谱分析将显示具有中间质量的部分氘化部分（40%+ 25%）的早期出现。在另一个极端，25%的溶菌酶分子可以在快相中折叠它们的a和P结构域。然后，质谱分析将显示早期形成的重级分（25%），其两个结构域都被保护并且完全氘代，早期形成的中间质量级分（15%），其仅α结构域被保护，以及未受保护的轻级分（60%），其直到过程后期才完全折叠。质谱结果明确选择第二种情况。此外，α结构域显然可以自身折叠，然后可以夹带或不夹带β B结构域，但β B结构域不能独立折叠。质谱实验具有普遍适用性。当任何两个结构元素在相同的时间尺度上折叠时，质谱可以指示这两个元素是在同一分子中一起折叠还是在不同分子中独立折叠。Jennings和Wright（2）使用HX脉冲标记实验和NMR分析来研究去血红素肌红蛋白（去除血红素基团的肌红蛋白分子）的折叠。结果定义了一种折叠中间体，其在小于5ms内形成，并且非常类似于先前已知的平衡折叠中间体，即所谓的熔融球形式的脱辅基肌红蛋白。这一结果将为越来越多的研究人员提供安慰和喜悦，他们试图通过研究可以直接以平衡形式获得和检查的折叠中间体来回避研究动力学折叠的问题。平衡熔融球的形式，它已被建议，提供了真正的动力学中间体容易获得的类似物。熔融球有一段有趣的历史。在经历了早期的怀疑之后，蛋白质化学家现在接受了这样一种观点，即蛋白质不仅可以以其天然和完全展开的状态存在，而且还可以以称为熔融球的中间形式存在。早期的熔融球是一个严格定义的结构，但不断增长的中间蛋白质形式的动物园已经扩大了定义，包括任何不完全天然但尚未完全展开的蛋白质形式。例子包括脱辅基肌红蛋白的平衡熔融球，它有三个正常的肌红蛋白螺旋，螺旋A，G和H，由...
S. Walter Englander for formation of the native structure. Miranker et al.(1) used these methods to study the folding of henegg lysozyme. Earlier hydrogen exchange (HX) labeling experiments showed that lysozyme shares a surprising and particularly frustrating as-pect of most folding behavior so far observed. Folding is heterogeneous. The rigorously purified protein preparation in the chemist's test tube splits into different subfractions that fold at different rates. This heterogeneity in folding leads to special problems of interpretation, which Miranker et al. solve by an ingenious applicaton of mass spectrometry. Mass spectrometry can separate the refolded proteins according to mass so that, unlike NMR analysis, the mo-lecular distribution of HD labeling obtained in an HX labeling experiment can be resolved. The single chain lysozyme molecule contains two distinct lobes: an a domain rich in a helix and a P domain rich in P sheet. Earlier work (6) showed that protec-tion against HX labeling develops in a fast phase (5 to 10 ms) for 40% of every measurable amide in the a domain and for 25% of every measurable amide in the P domain. The early folding phase might represent molecules with 40% having only their a domains folded and 25% only their P domains. In this case, a mass spectrometric analysis would show the early appearance of a partially deuterated fraction (40%+ 25%) with intermediate mass. At the other extreme, 25% of the lysozyme molecules may fold both their a and P domains in the fast phase. Then, a mass spectrometric analysis would display an early forming heavy fraction (25%) with both domains protected and fully deuterated, an early forming intermediate mass fraction (15%) with only the a domain protected, and an unprotected light fraction (60%) that does not fold at all until later in the process. The mass spectrometry results clearly select the second case. Furthermore, a domains apparently can fold by themselves and then may or may not entrain the, B domain, but, B domains do not fold independently. The mass spectrometry experiment hasgeneral applicability. When any two structural ele-ments fold on the same time scale, mass spectrometry can indicate whether the two elements fold together in the same molecule or independently in different molecules. Jennings and Wright (2) used the HX pulse labeling experiment with NMR analysis to study the folding of apomyoglobin, the myoglobin molecule with its heme group removed. The results define a folding intermediate that forms in less than 5 ms and strongly resembles a previously known (7) equilibrium folding intermediate, the so-called molten globule form of apomyoglobin. This result will provide comfort and joy to a growing group of investigators who have attempted to sidestep the problems of studying kinetic folding by studying folding intermediates that can be obtained and examined directly in an equilibrium form. Equilibrium molten globule forms, it has been suggested, provide easily accessible analogs of true kinetic intermediates. The molten globule has an interesting history. After some early skepticism, protein chemists have now embraced the idea that proteins can exist not only in their native and fully unfolded states but also as in-termediate forms referred to as molten globules. The molten globule in its early incarnation was a strictly defined construct (8), but a growing zoo of intermediate pro-tein forms hasbroadened the definition to include any protein form that is less than fully native but not yet fully unfolded. Examples include the equilibrium molten globule of apomyoglobin, which has three of the normal myoglobin helices, helices A, G, and H, identified by …