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首页> 外文期刊>Biochemistry >Kinetics of folding of the IgG binding domain of peptostreptococcal protein L.
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Kinetics of folding of the IgG binding domain of peptostreptococcal protein L.

机译：肽链球菌蛋白L的IgG结合域折叠的动力学。

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摘要

The kinetics of folding of a tryptophan containing mutant of the IgG binding domain of protein L were characterized using stopped-flow circular dichroism, stopped-flow fluorescence, and HD exchange coupled with high-resolution mass spectrometry. Both the thermodynamics and kinetics of folding fit well to a simple two-state model: (1) Guanidine induced equilibrium denaturation transitions measured by fluorescence and circular dichroism were virtually superimposable. (2) The kinetics of folding/unfolding were single exponential under all conditions examined, and the rate constants obtained using all probes were similar. (3) Mass spectra from pulsed HD exchange refolding experiments showed that a species with very little protection from exchange is converted to a fully protected species (the native state) at a rate very similar to that of the overall change in tryptophan fluorescence; no intervening partially protected species were observed. (4) Rate constants (in H2O) and m values for folding and unfolding determined by fitting observed relaxation rates obtained over a broad range of denaturant concentrations to a two-state model were consistent with the equilibrium parameters deltaG and m: -RT ln(k(u)/k(f))/deltaG(U)H2O = 1.02; (m(u) + m(f))/m = 1.08. In contrast to results with a number of other proteins, there was no deviation from linearity in plots of ln k(obs) versus guanidine at low guanidine concentrations, both in the presence and absence of 0.4 M Na2SO4, suggesting that significantly stabilized intermediates do not accumulate during folding. Although all of the change in fluorescence signal during folding in phosphate buffer was accounted for by the simple exponential describing the overall folding reaction, fluorescence-quenching experiments using sodium iodide revealed a small reduction in the extent of quenching of the protein within the first two milliseconds after initiation of refolding in low concentrations of guanidine, suggesting a partial collapse of the unfolded chain may occur under these conditions. Comparison with results on the structurally and functionally similar IgG binding domain of streptococcal protein G show intriguing differences in the folding of the two proteins.
机译：使用停止流圆二色性，停止流荧光和HD交换结合高分辨率质谱法，对蛋白L IgG结合域的含色氨酸突变体的折叠动力学进行了表征。折叠的热力学和动力学都很好地适合于简单的二态模型：（1）通过荧光和圆二色性测定的胍诱导的平衡变性转变实际上是可叠加的。 （2）在所有检查条件下，折叠/展开的动力学均为单指数，并且使用所有探针获得的速率常数相似。 （3）脉冲高清交换重折叠实验的质谱表明，一种几乎没有交换保护的物种会以与色氨酸荧光的整体变化非常相似的速率转化为完全保护的物种（天然状态）;没有观察到中间部分受保护的物种。 （4）通过将在宽范围的变性剂浓度范围内获得的观察到的弛豫率拟合到二态模型而确定的折叠和展开的速率常数（以H2O为单位）和m值与平衡参数delG和m：-RT ln（ k（u）/ k（f））/ deltaG（U）H2O = 1.02; （m（u）+ m（f））/ m = 1.08。与许多其他蛋白质的结果相反，在有和没有0.4 M Na2SO4的情况下，在低胍浓度下ln k（obs）对胍的图谱中的线性均无偏差，表明存在明显稳定的中间体在折叠过程中积累。尽管在磷酸盐缓冲液中折叠过程中荧光信号的所有变化都是通过描述整个折叠反应的简单指数来解决的，但是使用碘化钠进行的荧光猝灭实验显示，在头两毫秒内，蛋白质的猝灭程度有所降低在低浓度的胍开始重折叠后，表明在这些条件下未折叠链可能会部分折叠。与在链球菌蛋白G的结构和功能上相似的IgG结合结构域上的结果的比较表明，这两种蛋白的折叠具有令人感兴趣的差异。

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