FOLDING AND STABILITY OF A TRYPTOPHAN-CONTAINING MUTANT OF UBIQUITIN
FOLDING AND STABILITY OF A TRYPTOPHAN-CONTAINING MUTANT OF UBIQUITIN
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DOI：
10.1021/bi00078a034
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发表时间：
1993-07-13
期刊：
影响因子：
2.9
通讯作者：
RODER, H
中科院分区：
文献类型：
--
作者：
KHORASANIZADEH, S;PETERS, ID;RODER, H
为了提供用于泛素平衡和动力学折叠研究的荧光探针，使用大肠杆菌表达质粒中的盒式诱变来用色氨酸取代大部分埋藏的 Phe 45。在天然条件下，该 F45W 突变体的色氨酸荧光光谱在 336 nm 处表现出蓝移发射最大值，表明很大程度上是溶剂屏蔽的色氨酸环境。相比之下，6 M 盐酸胍 (GuHCl) 中的未折叠蛋白质在 353 nm 处显示出比游离色氨酸强 4 倍的发射带。 F45W 泛素的二维 H-1 NMR 谱是通过与已发表的野生型归属进行比较来确定的。该突变仅导致紧邻残基 45 的残基发生有限的化学位移变化。通过核 Overhauser 谱的初步分析证实了 F45W 与野生型泛素的结构相似性。 GuHCl 诱导的可逆去折叠转变的 NMR 和圆二色性测量表明，F45W 突变使折叠泛素结构的稳定性降低不到 0.4 kcal/mol。在与泛素激活酶 E1 的反应以及 ATP 依赖性蛋白质降解的体外测定中，发现突变体的生物活性与野生型没有区别。在多种 GuHCl 浓度 (0.5-6 M) 下，通过一系列荧光检测停流测量，在两个温度（8 和 25 摄氏度）下研究了 F45W 泛素的折叠和解折叠动力学。 25 摄氏度下的测量结果与二态模型一致，折叠和解折叠速率高于约 2 M GuHCl，具有强烈的变性依赖性。然而，在较低的变性剂浓度下，主要折叠相的速率变得与 GuHCl 无关，并且高达 60% 的总荧光变化发生在停流测量的 2 毫秒死区时间内。这些观察结果为在重折叠的最初几毫秒内形成早期折叠中间体提供了明确的证据，其中包含色氨酸 45 的部分发育的疏水核心。初始振幅的 S 型变性依赖性（表观中点为 1.3 M GuHCl）表明存在离散状态，该状态在较高变性剂浓度下不稳定。相比之下，没有证据表明 8 摄氏度的折叠动力学存在早期中间体。中间体在低温下的不稳定与主要通过疏水相互作用稳定的塌陷状态一致。
To provide a fluorescence probe for equilibrium and kinetic folding studies on ubiquitin, cassette mutagenesis in an Escherichia coli expression plasmid was used to replace the largely buried Phe 45 by a tryptophan. Under native conditions, the tryptophan fluorescence spectrum of this F45W mutant exhibits a blue-shifted emission maximum at 336 nm indicative of a largely solvent-shielded tryptophan environment. In contrast, the unfolded protein in 6 M guanidine hydrochloride (GuHCl) shows a 4-fold more intense emission band at 353 nm matching that of free tryptophan. The two-dimensional H-1 NMR spectrum of F45W ubiquitin was assigned by comparison with published assignments of the wild type. The mutation results in only limited chemical shift changes for residues in the immediate vicinity of residue 45. The structural similarity of F45W with wild-type ubiquitin was confirmed by a preliminary analysis of the nuclear Overhauser spectrum. NMR and circular dichroism measurements of the reversible GuHCl-induced unfolding transition show that the F45W mutation lowers the stability of the folded ubiquitin structure by less than 0.4 kcal/mol. The biological activity of the mutant was found to be indistinguishable from that of wild-type in terms of its reaction with the ubiquitin activating enzyme E1 and an in vitro assay of ATP-dependent protein degradation. The kinetics of folding and unfolding of F45W ubiquitin was studied at two temperatures (8 and 25-degrees-C) in a series of fluorescence-detected stopped-flow measurements over a wide range of GuHCl concentrations (0.5-6 M). The measurements at 25-degrees-C are consistent with a two-state model with strongly denaturant-dependent folding and unfolding rates above about 2 M GuHCl. However, at lower denaturant concentrations, the rate of the major folding phase becomes GuHCl-independent, and up to 60% of the total fluorescence change occurs during the 2-ms dead time of the stopped-flow measurement. These observations provide clear evidence for the formation of an early folding intermediate during the first few milliseconds of refolding with a partially developed hydrophobic core involving Trp 45. The sigmoid denaturant dependence of the initial amplitude with an apparent midpoint of 1.3 M GuHCl suggests the presence of a discrete state that is destabilized at higher denaturant concentrations. In contrast, there is no evidence for an early intermediate in the folding kinetics at 8-degrees-C. The destabilization of the intermediate at low temperature is consistent with a collapsed state stabilized primarily by hydrophobic interactions.