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Folding kinetics of the SH3 domain of PI3 kinase by real-time NMR combined with optical spectroscopy

机译：实时核磁共振-光谱结合PI3激酶SH3结构域的折叠动力学

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The refolding kinetics of the chemically denatured SH3 domain of phosphatidylinositol 3'-kinase (PI3-SH3) have been monitored by real-time one-dimensional H-1 NMR coupled with a variety of other biophysical techniques. These experiments indicate that the refolding kinetics of PI3-SH3 are biphasic. The slow phase (27 (+/-8)% amplitude) is due to a population of substantially unfolded molecules with an incorrectly configured cis proline residue. The fast phase (73 (+/-8)% amplitude) corresponds to the folding of protein molecules with proline residues in a tuans configuration in the unfolded state. NMR experiments indicate that the first species populated after the initiation of folding exhibit poor chemical shift dispersion and have spectra very similar to that of the denatured protein in 8 M guanidine hydrochloride. Linear combinations of the first spectrum and of the spectrum of the native protein accurately reconstruct all of the spectra acquired during refolding. Consistent with this, native side-chain and backbone H-alpha atom packing (NMR), secondary structure (far-UV circular dichroism), burial of aromatic residues (near-UV circular dichroism), intrinsic fluorescence and peptide binding activity are all recovered with effectively identical kinetics. Equilibrium unfolding and folding/unfolding kinetics yield,within experimental error, identical values for the free energy of unfolding (Delta G(u-H2O) = 3.38 kcal mol(-1)) and for the slope of the free energy of unfolding versus denaturant concentration (m(eq) = 2.33 kcal mol(-1) M-1). Together, these data provide strong evidence that PI3-SH3 folds without significant population of kinetic well-structured intermediates. That PI3-SH3 folds slowly (time constant 2.8 seconds in H2O at 20 degrees C) indicates that slow refolding is not always a consequence of kinetic traps but may be observed even when a protein appears to fold via a simple, two-state mechanism. (C) 1998 Academic Press Limited. [References: 56]
机译：磷脂酰肌醇3'-激酶（PI3-SH3）的化学变性SH3域的重折叠动力学已通过实时一维H-1 NMR和多种其他生物物理技术进行了监测。这些实验表明PI3-SH3的重折叠动力学是双相的。缓慢的相（振幅为27（+/- 8）％）是由于大量未折叠分子中顺式脯氨酸残基配置不正确所致。快相（73（+/- 8）％振幅）对应于处于未折叠状态的tuan构型中带有脯氨酸残基的蛋白质分子的折叠。 NMR实验表明，折叠开始后填充的第一个物种显示出较差的化学位移分散，并且具有与8 M盐酸胍中的变性蛋白质非常相似的光谱。第一光谱和天然蛋白质的光谱的线性组合准确地重建了在重折叠过程中获得的所有光谱。与此相一致，天然的侧链和主链H-α原子堆积（NMR），二级结构（远紫外圆二色性），掩埋芳香族残基（近紫外圆二色性），固有荧光和肽结合活性都得到了恢复。具有相同的动力学平衡展开和折叠/展开动力学的产量，在实验误差范围内，展开的自由能（Delta G（u-H2O）= 3.38 kcal mol（-1））以及展开自由度与变性剂的斜率相同浓度（m（eq）= 2.33 kcal mol（-1）M-1）。总之，这些数据提供了有力的证据，表明PI3-SH3折叠时没有大量的动力学结构良好的中间体。 PI3-SH3折叠缓慢（时间常数在20摄氏度的水中，时间常数为2.8秒）表明缓慢的重折叠并不总是由于动态陷阱而引起的，即使蛋白通过简单的两种状态机制折叠也可以观察到。 （C）1998 Academic Press Limited。 [参考：56]

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