Electron spin resonance in membrane research: protein-lipid interactions from challenging beginnings to state of the art.
Electron spin resonance in membrane research: protein-lipid interactions from challenging beginnings to state of the art.
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DOI：
10.1007/s00249-009-0512-3
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发表时间：
2010-03
影响因子：
2
通讯作者：
Marsh, Derek
中科院分区：
文献类型：
--
作者：
Marsh, Derek
关键词：
自旋标记在酰基链末端甲基端的脂质的常规电子顺磁共振（EPR）光谱能够从膜的流体双层区域中分离出直接与蛋白质接触的脂质。这样可以测定脂质-蛋白相互作用的化学计量学（即蛋白质周长的脂质位点数量）和蛋白质对不同脂质种类的选择性（即相对于背景脂质的关联常数）。自旋标签EPR数据总结了20或更多不同的跨膜肽和蛋白质，以及7种不同的脂质。双组分常规自旋标记EPR光谱的线形模拟允许估计蛋白质相关脂质与膜的散装流体区域中的脂质交换的速率。对于对蛋白质没有选择性的脂质，交换的固有速率在10 MHz范围内：比流体脂质膜中的扩散交换速率慢10倍。相对于背景脂质，对蛋白质有亲和力的脂质，离开蛋白质的速率相应较慢。非线性EPR依赖于高辐射强度下光谱的饱和，对自旋晶格弛豫时间尺度（即微秒级）上的动力学最敏感。渐进式饱和和饱和转移EPR实验都提供了明确的证据，证明蛋白质界面上的脂质在这个时间尺度上进行交换。非线性EPR对低频自旋交换的敏感性也允许在双标记实验中定位自旋标记的膜蛋白残基相对于自旋标记的脂质的残基。
Conventional electron paramagnetic resonance (EPR) spectra of lipids that are spin-labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane. This allows determination of both the stoichiometry of lipid–protein interaction (i.e., number of lipid sites at the protein perimeter) and the selectivity of the protein for different lipid species (i.e., association constants relative to the background lipid). Spin-label EPR data are summarised for 20 or more different transmembrane peptides and proteins, and 7 distinct species of lipids. Lineshape simulations of the two-component conventional spin-label EPR spectra allow estimation of the rate at which protein-associated lipids exchange with those in the bulk fluid regions of the membrane. For lipids that do not display a selectivity for the protein, the intrinsic off-rates for exchange are in the region of 10 MHz: less than 10× slower than the rates of diffusive exchange in fluid lipid membranes. Lipids with an affinity for the protein, relative to the background lipid, have off-rates for leaving the protein that are correspondingly slower. Non-linear EPR, which depends on saturation of the spectrum at high radiation intensities, is optimally sensitive to dynamics on the timescale of spin-lattice relaxation, i.e., the microsecond regime. Both progressive saturation and saturation transfer EPR experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale. The sensitivity of non-linear EPR to low frequencies of spin exchange also allows the location of spin-labelled membrane protein residues relative to those of spin-labelled lipids, in double-labelling experiments.
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影响因子：
2.9
作者：
Anbazhagan, V.;Vijay, N.;Marsh, D.
通讯作者：
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作者：
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作者：
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通讯作者：
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