Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing.
Early events during folding of wild-type staphylococcal nuclease and a single-tryptophan variant studied by ultrarapid mixing.
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DOI：
10.1016/j.jmb.2004.02.044
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发表时间：
2004-04
影响因子：
5.6
通讯作者：
K. Maki;Hong Cheng;D. A. Dolgikh;M. Shastry;Heinrich Roder;Heinrich Roder
中科院分区：
文献类型：
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作者：
K. Maki;Hong Cheng;D. A. Dolgikh;M. Shastry;Heinrich Roder;Heinrich Roder
采用死时间为100μ的连续流动混合装置，结合内源性色氨酸和1-苯基-8-苯基-8-磺酸(ANS)荧光，研究了葡萄球菌核酸酶(SNase)折叠早期的结构形成过程。用His取代野生型β酶中的单一Trp140，并结合Phe76的Trp替换，获得了一个在N端Trp76SNase中具有唯一色氨酸荧光团的突变体。选择了P47G、P117G和H124L突变的共同背景，以稳定蛋白质并防止顺式脯氨酸异构体在自然条件下的积累。WT∗SNase在限速折叠步骤(∼100ms)之前没有显示色氨酸荧光变化，而F76W/W140H变体在早期折叠阶段显示额外的变化(增强)，时间常数为75μS。两种蛋白质都显示出ANS荧光的显著增加和这一早期折叠事件的相同速率。这些发现与快速积累的状态相一致，该组合包含一个松散排列的疏水核心，主要涉及β-Barrel结构域，而涉及Trp140的α-螺旋结构域中的特定相互作用仅在折叠的最后阶段形成。这两个变体表现出相同数量的动力学相和非常相似的速率，这一事实证实了折叠机制没有受到F76W/W140H突变的干扰。然而，第76位的Trp报告了在N-末端β-Sheet区域快速形成疏水簇，而野生型Trp140在折叠的早期阶段是沉默的。对这两种蛋白质的非折叠动力学和热力学随尿素浓度变化的定量模拟表明，F76W/W140H突变选择性地破坏了相对于WT∗SNase的天然状态的稳定，而瞬时中间产物的稳定性保持不变，导致中间产物在中等变性剂浓度的平衡条件下积累。
A continuous-flow mixing device with a dead time of 100 μs coupled with intrinsic tryptophan and 1-anilinonaphthalene-8-sulfonate (ANS) fluorescence was used to monitor structure formation during early stages of the folding of staphylococcal nuclease (SNase). A variant with a unique tryptophan fluorophore in the N-terminal β-barrel domain (Trp76 SNase) was obtained by replacing the single Trp140 in wild-type SNase with His in combination with Trp substitution of Phe76. A common background of P47G, P117G and H124L mutations was chosen in order to stabilize the protein and prevent accumulation of cis proline isomers under native conditions. In contrast to WT∗SNase, which shows no changes in tryptophan fluorescence prior to the rate-limiting folding step (∼100 ms), the F76W/W140H variant shows additional changes (enhancement) during an early folding phase with a time constant of 75 μs. Both proteins exhibit a major increase in ANS fluorescence and identical rates for this early folding event. These findings are consistent with the rapid accumulation of an ensemble of states containing a loosely packed hydrophobic core involving primarily the β-barrel domain while the specific interactions in the α-helical domain involving Trp140 are formed only during the final stages of folding. The fact that both variants exhibit the same number of kinetic phases with very similar rates confirms that the folding mechanism is not perturbed by the F76W/W140H mutations. However, the Trp at position 76 reports on the rapid formation of a hydrophobic cluster in the N-terminal β-sheet region while the wild-type Trp140 is silent during this early stage of folding. Quantitative modeling of the (un)folding kinetics and thermodynamics of these two proteins versus urea concentration revealed that the F76W/W140H mutation selectively destabilizes the native state relative to WT∗SNase while the stability of transient intermediates remains unchanged, leading to accumulation of intermediates under equilibrium conditions at moderate denaturant concentrations.