Sparse Labeling PELDOR Spectroscopy on Multimeric Mechanosensitive Membrane Channels.
Sparse Labeling PELDOR Spectroscopy on Multimeric Mechanosensitive Membrane Channels.
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DOI：
10.1016/j.bpj.2017.09.005
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发表时间：
2017-11-07
影响因子：
3.4
通讯作者：
Bode BE
中科院分区：
文献类型：
--
作者：
Ackermann K;Pliotas C;Valera S;Naismith JH;Bode BE
脉冲电子顺磁共振(EPR)正被应用于由多个亚单位组成的更复杂的生物系统。随着PULSE EPR报告天然环境中不需要结晶的重要但不同的构象状态，膜通道蛋白引起了人们的极大兴趣。脉冲电子顺磁共振，以脉冲电子-电子双共振(PELDOR)的形式，使用定点自旋标记，最常被用来精确地确定结构不同区域之间的距离(在纳米范围内)。然而，由于扭曲的多自旋效应，PELDOR数据分析在包含两个以上自旋的系统(例如，同源多聚体)中更具挑战性。如果不抑制这些影响，PELDOR数据中包含的许多信息就不能可靠地检索出来。因此，对于未来PELDOR在结构生物学中的应用来说，开发合适的方法来克服多自旋问题是至关重要的。在这里，两种不同的方法来抑制PELDOR中的多自旋效应，稀疏标记蛋白质(降低标记效率f)和降低自旋的激发几率(λ)，在两个不同的细菌机械敏感通道上进行了比较。对于大肠杆菌的大电导五聚体通道(MSCL)和小电导七聚体通道(MSCS)，我们测试了胞液或跨膜区含有自旋标记的突变体。数据表明，与标准的PELDOR测量相比，这两种方法都可以显著改善距离分布，并证实需要λ；lt；1/(n−1)来充分抑制多自旋效应(n是系统中的自旋数量)。稀疏标记方法对于胞质突变体具有明显的优势，因为其敏感度损失明显较小。对于跨膜突变体，这一优势不那么明显，但对MSCs仍然有用，但MSCL的性能较差，可能是由于较大的抗磁性自旋标记类似物的结构扰动。
Pulse electron paramagnetic resonance (EPR) is being applied to ever more complex biological systems comprising multiple subunits. Membrane channel proteins are of great interest as pulse EPR reports on functionally significant but distinct conformational states in a native environment without the need for crystallization. Pulse EPR, in the form of pulsed electron-electron double resonance (PELDOR), using site-directed spin labeling, is most commonly employed to accurately determine distances (in the nanometer range) between different regions of the structure. However, PELDOR data analysis is more challenging in systems containing more than two spins (e.g., homomultimers) due to distorting multispin effects. Without suppression of these effects, much of the information contained in PELDOR data cannot be reliably retrieved. Thus, it is of utmost importance for future PELDOR applications in structural biology to develop suitable approaches that can overcome the multispin problem. Here, two different approaches for suppressing multispin effects in PELDOR, sparse labeling of the protein (reducing the labeling efficiency f) and reducing the excitation probability of spins (λ), are compared on two distinct bacterial mechanosensitive channels. For both the pentameric channel of large conductance (MscL) and the heptameric channel of small conductance (MscS) of Escherichia coli, mutants containing a spin label in the cytosolic or the transmembrane region were tested. Data demonstrate that distance distributions can be significantly improved with either approach compared to the standard PELDOR measurement, and confirm that λ < 1/(n−1) is needed to sufficiently suppress multispin effects (with n being the number of spins in the system). A clear advantage of the sparse labeling approach is demonstrated for the cytosolic mutants due to a significantly smaller loss in sensitivity. For the transmembrane mutants, this advantage is less pronounced but still useful for MscS, but performance is inferior for MscL possibly due to structural perturbations by the bulkier diamagnetic spin label analog.
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旋转对称的同性恋者中的Peldor。
影响因子：
1.7
作者：
Giannoulis A;Ward R;Branigan E;Naismith JH;Bode BE
通讯作者：
Bode BE
影响因子：
1
作者：
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通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
2.2
作者：
Chiang, YW;Borbat, PP;Freed, JH
通讯作者：
Freed, JH
影响因子：
3.3
作者：
Jeschke, Gunnar;Sajid, Muhammad;Godt, Adelheid
通讯作者：
Godt, Adelheid