Revealing conformational substates of lipidated N-Ras protein by pressure modulation
Revealing conformational substates of lipidated N-Ras protein by pressure modulation
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DOI：
10.1073/pnas.1110553109
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发表时间：
2012-01-10
影响因子：
11.1
通讯作者：
Winter, Roland
中科院分区：
文献类型：
--
作者：
Kapoor, Shobhna;Triola, Gemma;Winter, Roland
蛋白质功能的调节通常与由化学或物理信号触发的构象转换有关。为了评估这种构象变化，并阐明后续蛋白质功能的潜在分子机制，实验鉴定的构象substates和表征的构象平衡是强制性的。我们应用压力调制结合FTIR光谱，以揭示光谱解析的substates之间的平衡，过时的信号蛋白N-Ras。压力的优点是它的热力学共轭是体积，一个与结构直接相关的参数。通过压力扰动来探测在模型生物膜存在和不存在的情况下N-Ras在不同核苷酸结合状态下的构象动力学。我们发现，不仅核苷酸结合，而且膜的存在下有一个剧烈的影响的构象动力学和选择的构象substates的蛋白质，和一个新的substate后出现的膜结合可能会被发现。这个新的亚态的人口是伴随着G结构域的结构重新取向，也表明由互补的ATR-FTIR和IRRAS测量。因此，这些研究结果表明，膜控制信号传导构象作为一个有效的相互作用的合作伙伴，这有后果的G-结构域的取向膜相关的N-Ras，这反过来又被称为是至关重要的效应器和调制器的相互作用。最后，这些结果提供了深入了解压力的影响，在极端环境条件下生活的生物体Ras控制的信号事件，因为他们遇到的深海压力达到千巴范围。
Regulation of protein function is often linked to a conformational switch triggered by chemical or physical signals. To evaluate such conformational changes and to elucidate the underlying molecular mechanisms of subsequent protein function, experimental identification of conformational substates and characterization of conformational equilibria are mandatory. We apply pressure modulation in combination with FTIR spectroscopy to reveal equilibria between spectroscopically resolved substates of the lipi-dated signaling protein N-Ras. Pressure has the advantage that its thermodynamic conjugate is volume, a parameter that is directly related to structure. The conformational dynamics of N-Ras in its different nucleotide binding states in the absence and presence of a model biomembrane was probed by pressure perturbation. We show that not only nucleotide binding but also the presence of the membrane has a drastic effect on the conformational dynamics and selection of conformational substates of the protein, and a new substate appearing upon membrane binding could be uncovered. Population of this new substate is accompanied by structural reorientations of the G domain, as also indicated by complementary ATR-FTIR and IRRAS measurements. These findings thus illustrate that the membrane controls signaling conformations by acting as an effective interaction partner, which has consequences for the G-domain orientation of membrane-associated N-Ras, which in turn is known to be critical for its effector and modulator interactions. Finally, these results provide insights into the influence of pressure on Ras-controlled signaling events in organisms living under extreme environmental conditions as they are encountered in the deep sea where pressures reach the kbar range.