Use of the novel fluorescent amino acid ρ-cyanophenylalanine offers a direct probe of hydrophobic core formation during the folding of the n-terminal domain of the ribosomal protein L9 and provides evidence for two-state folding
Use of the novel fluorescent amino acid ρ-cyanophenylalanine offers a direct probe of hydrophobic core formation during the folding of the n-terminal domain of the ribosomal protein L9 and provides evidence for two-state folding
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DOI：
10.1021/bi7010674
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发表时间：
2007-10-30
期刊：
影响因子：
2.9
通讯作者：
Raleighj, Daniel P.
中科院分区：
文献类型：
--
作者：
Aprilakis, Konstantinos N.;Taskent, Humeyra;Raleighj, Daniel P.
荧光检测停流测量是研究蛋白质折叠动力学的首选方法。然而，该方法受到的限制是，感兴趣的蛋白质必须含有内在的荧光团，或者可以耐受通过突变引入的荧光团。最近，苯丙氨酸的氰基(腈)类似物被提出用作荧光类似物。在这里，我们利用这一新的方法学来监测L9(NTL9)N-末端结构域折叠过程中疏水核心的形成。用对氰基苯丙氨酸(p-氰基-Phe)取代了完全处于NTL9折叠状态的苯丙氨酸5。该衍生物报告了疏水核心的形成。该变异体采用与野生型NTL9相同的折叠，略有更稳定。用盐酸胍和尿素跳跃实验监测复性和去折叠。在这两种情况下，观察到的松弛速率与变性剂浓度的自然对数的曲线图，即所谓的人字形曲线图，显示出两态折叠所预期的特征V形，并且在低变性剂浓度下没有观察到偏离线性的迹象。由测量的折叠和展开速率计算的稳定性与由平衡测量得到的值以及m值非常吻合。由tanford参数定义的折叠过渡态的相对紧密度与野生型的相同。这些结果说明了p-氰基-Phe类似物在蛋白质折叠研究中的适用性，并为NTL9的两态折叠提供了进一步的证据。
Fluorescence-detected stopped flow measurements are the method of choice for studies of protein folding kinetics. However, the methodology suffers from the limitation that the protein of interest either must contain an intrinsic fluorophore or can tolerate its introduction by mutagenesis. Recently, the cyano (nitrile) analogue of phenylalanine has been proposed for use as a fluorescence analogue. Here we take advantage of this new methodology to monitor the formation of the hydrophobic core during the folding of the N-terminal domain of L9 (NTL9). Phenylalanine 5, which is completely buried in the folded state of NTL9, was replaced with p-cyanophenylalanine (p-cyano-Phe). This derivative reports on the formation of the hydrophobic core. The variant adopts the same fold as wild-type NTL9 and is slightly more stable. Refolding and unfolding were monitored using both guanidine HC1 and urea jump experiments. In both cases, plots of the natural log of the observed relaxation rate versus denaturant concentration, so-called chevron plots, exhibited the characteristic V shape expected for two-state folding, and no hint of deviation from linearity was observed at low denaturant concentrations. The stability calculated from the measured folding and unfolding rates is in very good agreement with the value obtained from equilibrium measurements as is the m value. The relative compactness of the transition state for folding as defined by the Tanford parameter is identical to that of the wild type. The results illustrate the applicability of p-cyano-Phe analogues in protein folding studies and provide further evidence of two-state folding of NTL9.