Molecular force modulation spectroscopy revealing the dynamic response of single bacteriorhodopsins
Molecular force modulation spectroscopy revealing the dynamic response of single bacteriorhodopsins
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DOI：
10.1529/biophysj.104.052746
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发表时间：
2005-02-01
影响因子：
3.4
通讯作者：
Humphris, ADL
中科院分区：
文献类型：
--
作者：
Janovjak, H;Müller, DJ;Humphris, ADL
原子力显微镜的最新进展允许球状和膜蛋白在单分子水平上机械展开。提出的是现有的力谱实验的扩展。当从天然紫色膜展开单个细菌视紫红质时，以3 kHz的频率向悬臂的垂直位移提供小的振荡幅度（6 - 9 nm）。通过对蛋白质-蛋白质系统的相位和振幅响应进行转换，揭示了弹性（保守）和粘性（耗散）对展开过程的贡献。延伸部分的蛋白质的弹性响应（刚度）在几十pN/nm的范围内，可以很好地描述的蠕虫状链模型的衍生物。在粘性响应的离散事件正好与单一的二级结构元素的展开，并在1 μ Ns/m的范围内。此外，这些力调制光谱实验揭示了新的机械展开中间体的细菌视紫红质。我们发现，扭结导致跨膜螺旋的展开协同性的损失。重建力-距离光谱的振幅-距离光谱的整合验证了他们的位置，提供了一种新的方法来检测中间体在强制解折叠的单个蛋白质。
Recent advances in atomic force microscopy allowed globular and membrane proteins to be mechanically unfolded on a single-molecule level. Presented is an extension to the existing force spectroscopy experiments. While unfolding single bacteriorhodopsins from native purple membranes, small oscillation amplitudes ( 6 - 9 nm) were supplied to the vertical displacement of the cantilever at a frequency of 3 kHz. The phase and amplitude response of the cantilever-protein system was converted to reveal the elastic ( conservative) and viscous ( dissipative) contributions to the unfolding process. The elastic response ( stiffness) of the extended parts of the protein were in the range of a few tens pN/nm and could be well described by the derivative of the wormlike chain model. Discrete events in the viscous response coincided with the unfolding of single secondary structure elements and were in the range of 1 muNs/m. In addition, these force modulation spectroscopy experiments revealed novel mechanical unfolding intermediates of bacteriorhodopsin. We found that kinks result in a loss of unfolding cooperativity in transmembrane helices. Reconstructing force-distance spectra by the integration of amplitude-distance spectra verified their position, offering a novel approach to detect intermediates during the forced unfolding of single proteins.