Probing local structural fluctuations in myoglobin by size-dependent thiol-disulfide exchange.
Probing local structural fluctuations in myoglobin by size-dependent thiol-disulfide exchange.
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通过尺寸依赖性硫醇-二硫化物交换探测肌红蛋白的局部结构波动。
DOI：
10.1002/pro.440
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Loh,StewartN
中科院分区：
文献类型：
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作者：
Stratton,MargaretM;Cutler,ThomasA;Ha,Jeung-Hoi;Loh,StewartN
所有的蛋白质都会经历局部结构波动（LSF）或呼吸运动。这些运动可能对功能很重要，但人们对此了解甚少。LSFs最初被定义为开放事件的酰胺氢交换（HX）实验，其中暴露了少量的骨干酰胺到1H/2H交换，但其交换率是独立的变性剂浓度。在这里，我们使用大小依赖性巯基-二硫键交换（SX）来表征肌红蛋白（Mb）的单个含半胱氨酸变体中的LSF。SX通过提供关于破坏侧链包装相互作用的运动的信息来补充HX。最重要的是，不同尺寸和化学性质的探针试剂可用于表征结构打开事件的尺寸和打开状态的性质。我们使用硫代磺酸盐试剂（126 - 274 Da）来调查Cys残基的进入，Cys残基被埋在Mb中特定的螺旋包装界面处。在每种情况下，开放的自由能与探针试剂的回转半径线性增加。斜率和截距被解释为产生关于暴露掩埋的硫醇基团的开口事件的大小的信息。斜率参数在测试的Cys位置之间变化超过10倍，表明呼吸运动的大小在整个蛋白质中变化很大。我们的研究结果为长期存在的问题提供了见解：蛋白质在其天然状态下的刚性或柔性如何？
All proteins undergo local structural fluctuations (LSFs) or breathing motions. These motions are likely to be important for function but are poorly understood. LSFs were initially defined by amide hydrogen exchange (HX) experiments as opening events, which expose a small number of backbone amides to1H/2H exchange, but whose exchange rates are independent of denaturant concentration. Here, we use size‐dependent thiol‐disulfide exchange (SX) to characterize LSFs in single cysteine‐containing variants of myoglobin (Mb). SX complements HX by providing information on motions that disrupt side chain packing interactions. Most importantly, probe reagents of different sizes and chemical properties can be used to characterize the size of structural opening events and the properties of the open state. We use thiosulfonate reagents (126–274 Da) to survey access to Cys residues, which are buried at specific helical packing interfaces in Mb. In each case, the free energy of opening increases linearly with the radius of gyration of the probe reagent. The slope and the intercept are interpreted to yield information on the size of the opening events that expose the buried thiol groups. The slope parameter varies by over 10‐fold among Cys positions tested, suggesting that the sizes of breathing motions vary substantially throughout the protein. Our results provide insight to the longstanding question: how rigid or flexible are proteins in their native states?