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Characterization of DLC1-SAM Equilibrium Unfolding at the Amino Acid Residue Level

机译：氨基酸残基水平上DLC1-SAM平衡展开的表征

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摘要

Sterile α motif (SAM) domains are found in many different proteins and shown to play important roles in various biological processes. The N-terminal domain of deleted in liver cancer 1 (DLC1) protein is a SAM domain which exists in a monomeric form in aqueous solution and facilitates the distribution of EF1A1 to the membrane periphery and ruffles upon growth factor stimulation. Here, we report the structure of an N-terminal truncated DLC1 SAM domain (DLC1-SAM) and its urea-induced equilibrium unfolding investigated with various biophysical methods such as CD, fluorescence emission spectroscopy, and NMR. CD and tryptophan intrinsic fluorescence emission data imply that the unfolding of DLC1-SAM follows a simple two-state mechanism, yet the NMR data suggest the presence of at least one intermediate state. The intermediate cannot be detected by NMR, but it does not exist in large aggregates as shown by analytical ultracentrifugation experiments. Analysis of the free energy values for different residues shows that in the transition from the native state to non-native states the C-terminal helix is somewhat more stable than the other parts of the protein, whereas in the transition from the native and intermediate states to the denatured state, the stabilities of different residues are similar except for that of the region surrounding residues D37−F40 which has lower stability and is more readily denatured at high urea concentrations. Analysis of the midpoints of the transitions shows that the unfolding of the native state and formation of the denatured state are not cooperative and the unfolding of only a few residues seems to follow a two-state mechanism.
机译：在许多不同的蛋白质中发现了不育的α基序（SAM）结构域，并显示了它们在各种生物过程中的重要作用。肝癌1（DLC1）蛋白缺失的N末端结构域是SAM结构域，其以单体形式存在于水溶液中，并有助于EF1A1分布到膜周围并在生长因子刺激下起皱。在这里，我们报告的N末端截短DLC1 SAM域（DLC1-SAM）的结构及其脲诱导的平衡展开，已通过各种生物物理方法（例如CD，荧光发射光谱和NMR）进行了研究。 CD和色氨酸固有的荧光发射数据暗示DLC1-SAM的展开遵循简单的两个状态机制，而NMR数据表明存在至少一个中间状态。中间体无法通过NMR检测到，但不存在于大型聚集体中，如分析超离心实验所示。对不同残基的自由能值的分析表明，在从天然状态到非天然状态的转变中，C末端螺旋比蛋白质的其他部分更稳定，而在从天然状态和中间状态的转变中在变性状态下，不同残基的稳定性相似，但残基D37-F40周围区域的稳定性较低，在高尿素浓度下更容易变性。对转变中点的分析表明，天然状态的展开和变性状态的形成是不合作的，只有少数残基的展开似乎遵循两种状态的机制。

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  • 来源
    《Biochemistry》 |2009年第19期|p.4040-4049|共10页
  • 作者

    Shuai Yang Christian G. Noble and Daiwen Yang;

  • 作者单位

    ‡ Department of Chemistry, 3 Science Drive 3, Faculty of Science, National University of Singapore, Singapore 117543§ Department of Biological Sciences, 14 Science Drive 4, Faculty of Science, National University of Singapore, Singapore 117543 Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673;

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