Equilibrium unfolding of yeast phosphoglycerate kinase and its mutants lacking one or both native tryptophans: a circular dichroism and steady-state and time-resolved fluorescence study.
Equilibrium unfolding of yeast phosphoglycerate kinase and its mutants lacking one or both native tryptophans: a circular dichroism and steady-state and time-resolved fluorescence study.
复制标题
酵母磷酸甘油酸激酶及其缺乏一种或两种天然色氨酸的突变体的平衡展开：圆二色性以及稳态和时间分辨荧光研究。
DOI：
10.1021/bi00174a031
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发表时间：
1994
期刊：
影响因子：
2.9
通讯作者：
Mas,MT
中科院分区：
文献类型：
--
作者：
Szpikowska,BK;Beechem,JM;Sherman,MA;Mas,MT
摘要：酵母3-磷酸甘油激酶含有两个位于羧基末端的色氨酸，以及7个酪氨酸，5个位于氨基末端，1个位于结构域界面，1个位于羧基末端。通过用苯丙氨酸取代天然色氨酸W308和W333，利用位点特异性诱变技术构建了两个单色氨酸突变体和一个无色氨酸突变体。突变已被证明对突变体的整体结构和酶特性有相对较小的影响。两种色氨酸都在折叠状态下熄灭。单色氨酸突变体和野生型蛋白的稳态发射光谱和色氨酸量子产率是相同的。在展开时观察到色氨酸发射最大值和稳态发射强度的巨大变化。远紫外圆二色性和稳态以及时间分辨荧光光谱已被用于监测这些突变体和野生型PGK的平衡展开转变。对于每种蛋白质，CD和稳态荧光的转变几乎是一致的，这表明局部荧光探针监测的结构变化和对整体结构变化敏感的椭圆率变化报告了单一的合作转变，符合两态展开机制。两种色氨酸都有三个生命周期，它们遵循一个类似的模式，作为变性剂浓度的函数。与两个较长寿命相关的振幅项随着展开而增加，而短寿命振幅则减小。因此，这些种群振幅似乎分别代表了展开状态和折叠状态的标记。由各幅值项分析所得的过渡中点与由稳态总强度变化所得的过渡中点一致。相反，由寿命变化和与长相关时间相关的指数前变化确定的衰减似乎在稳态强度变化和CD之后的转变之前。与WT、W308和W333相关的时间分辨各向异性衰减在折叠的蛋白质中是不同的，在未折叠的蛋白质中基本相同。
Revised Manuscript Received January 4, 1994Ū abstract: Yeast 3-phosphoglycerate kinase contains two tryptophans, both situated in the carboxy-terminal domain, and seven tyrosines, five in the amino-terminal domain, one in the domain-domain interface, and one in the carboxy-terminal domain. Site-specific mutagenesis has been used to construct two single-tryptophan mutants and one no-tryptophan mutant by replacing one or both native tryptophans, W308 and W333, with phenylalanines. The mutations have been shown to have a relatively small effect on the overall structure and enzymatic properties of the mutants. Both tryptophans are quenched in the folded state. The steady-state emission spectra and tryptophan quantum yields are the same in the single-tryptophan mutants and in the wild-type protein. Large changes in the tryptophan emission maxima and steady-state emission intensities are observed upon unfolding. Far-UV circular dichroism and steady-state as well as time-resolved fluorescence spectroscopy have been used to monitor the equilibrium unfolding transitions of these mutants and wild-type PGK. For each protein, the transitions followed by CD and steady-state fluorescence are nearly coincident, suggesting that the structural changes monitored by local fluorescence probes and ellipticity changes, which are sensitive to the changes in the overall structure, report a single cooperative transition, consistent with a two-state unfolding mechanism. Both tryptophans have three lifetimes, which follow a similar pattern as a function of denaturant concentration. The amplitude termsassociated with the two longer lifetimes increase with unfolding while the short lifetime amplitude decreases. It thus appears that these population amplitudes represent markers for the unfolded and folded states, respectively. The transition midpoints calculated from the analysis of each amplitude term are identical with those determined from the steady-state total intensity changes. In contrast, those determined from the lifetime changes and from the preexponential associated with the long correlation time appear to precede the transitions followed by steady-state intensity changes and CD. The time-resolved anisotropy decays associated with WT, W308, and W333 are distinct in the folded proteinsand become essentially identical in the unfolded state.