Direct characterization of the folded, unfolded and urea-denatured states of the C-terminal domain of the ribosomal protein L9.
Direct characterization of the folded, unfolded and urea-denatured states of the C-terminal domain of the ribosomal protein L9.
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核糖体蛋白 L9 C 端结构域的折叠、未折叠和尿素变性状态的直接表征。
DOI：
10.1016/j.jmb.2005.04.017
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
Raleigh,DanielP
中科院分区：
文献类型：
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作者：
Li,Ying;Picart,Francis;Raleigh,DanielP
核糖体蛋白 L9 (CTL9) 的分离 C 端结构域的稳定性强烈依赖于 pH 值。 pH 值低于 4.2 时，折叠和展开状态均显着增多。它们的相互转化在核磁共振化学位移时间尺度上很慢，并且观察到来自每个状态的单独的、分辨率良好的共振。这使得可以通过使用脉冲场梯度核磁共振实验在相同条件下研究两种状态的流体动力学特性。已得出折叠、未折叠和尿素变性蛋白质分子在 pD 3.8 时的流体动力学半径。与尿素变性蛋白（pD 3.8 时的 33.6Å）相比，酸变性蛋白的流体动力学半径明显更小，为 28.2Å。远紫外圆二色光谱表明，酸变性组中比尿素变性组中保留了更多的残余二级结构。 ANS 结合实验和 CD 数据分析表明，这种酸变性物质不是熔球状态。 CTL9 的扩散测量在 2.1 至 7.0 的 pD 范围内进行。折叠和酸解折叠蛋白质的流体动力学半径在 pD 4 以下开始增加，酸解折叠状态的水合半径从 pD 4.2 时的 25.1Å 增加到 pD 2.1 时的 33.5Å。尿素变性蛋白质的流体动力学半径对 pH 值的敏感度要低得多。 pD 2.1（不含尿素）的未折叠蛋白质与 pD 3.8 的尿素变性蛋白质具有几乎相同的流体动力学半径。然而，圆二色光谱显示残余二级结构存在显着差异，并且酸变性状态包含更多结构。
The stability of the isolated C-terminal domain of the ribosomal protein L9 (CTL9) is strongly dependent upon pH. Below pH 4.2, the folded and unfolded states are both populated significantly. Their interconversion is slow on the NMR chemical shift time-scale and separate, well-resolved resonances from each state are observed. This allows the hydrodynamic properties of both states to be studied under identical conditions by using pulse field gradient NMR experiments. Hydrodynamic radii of the folded, unfolded and urea denatured protein molecules at pD 3.8 have been derived. The acid-denatured protein has a significantly smaller hydrodynamic radius, 28.2Å, compared to that of the urea-denatured protein, which is 33.6Å at pD 3.8. Far-UV CD spectra show that there is more residual secondary structure retained in the acid-denatured ensemble than in the urea-denatured one. ANS binding experiments and analysis of the CD data show that this acid-denatured species is not a molten globule state. Diffusion measurements of CTL9 were conducted over the pD range from 2.1 to 7.0. The hydrodynamic radii of both the folded and the acid-unfolded protein start to increase below pD 4, with the radius of hydration of the acid-unfolded state increasing from 25.1Å at pD 4.2 to 33.5Å at pD 2.1. The hydrodynamic radius of the urea-denatured protein is much less sensitive to pH. The unfolded protein at pD 2.1, no urea, has almost the same hydrodynamic radius as the urea-denatured protein at pD 3.8. The CD spectra, however, show significant differences in residual secondary structure, and the acid-denatured state contains more structure.