CD spectroscopy of peptides and proteins bound to large unilamellar vesicles.
CD spectroscopy of peptides and proteins bound to large unilamellar vesicles.
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DOI：
10.1007/s00232-010-9291-0
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发表时间：
2010-08
影响因子：
2.4
通讯作者：
White, Stephen H.
中科院分区：
文献类型：
--
作者：
Ladokhin, Alexey S.;Fernandez-Vidal, Monica;White, Stephen H.
关键词：
圆二色性（CD）光谱是测定膜中蛋白质和多肽构象的重要工具。它对于测量多肽进入脂质囊泡的自由能特别有用。人们普遍认为，这种CD测量只能使用超声小单层囊泡（suv）进行，因为与挤压大单层囊泡（LUVs）相关的光散射高得令人无法接受。我们用几种实验方法研究了这个问题，其中手性物体（即肽或蛋白质）放置在膜上和膜外。我们证明了在luv存在的情况下可以收集精确的CD光谱。这一点很重要，因为suv与luv不同，是亚稳态的，因此不适合平衡热力学测量。我们的数据表明，在高达3mm的luv存在下，肽的未扭曲CD光谱可以在波长超过200 nm的地方测量，在高达7mm的luv存在下，可以在波长超过215 nm的地方测量。我们介绍了一种表征任意直径的囊泡悬浮液对CD光谱的光散射和吸收影响的简单方法。以蜂毒蛋白为例，我们证明了CD光谱可以用来测定luv中肽的螺旋分数含量，并测量它们分裂成luv的自由能。
Circular dichroism (CD) spectroscopy is an essential tool for determining the conformation of proteins and peptides in membranes. It can be particularly useful for measuring the free energy of partitioning of peptides into lipid vesicles. The belief is broadly held that such CD measurements can only be made using sonicated small unilamellar vesicles (SUVs) because light scattering associated with extruded large unilamellar vesicles (LUVs) is unacceptably high. We have examined this issue using several experimental approaches in which a chiral object (i.e., peptide or protein) is placed both on the membrane and outside the membrane. We show that accurate CD spectra can be collected in the presence of LUVs. This is important because SUVs, unlike LUVs, are metastable and consequently unsuitable for equilibrium thermodynamic measurements. Our data reveal that undistorted CD spectra of peptides can be measured at wavelengths above 200 nm in the presence of up to 3 mM LUVs and above 215 nm in the presence of up to 7 mM LUVs. We introduce a simple way of characterizing the effect on CD spectra of light scattering and absorption arising from suspensions of vesicles of any diameter. Using melittin as an example, we show that CD spectroscopy can be used to determine the fractional helical content of peptides in LUVs and to measure their free energy of partitioning of into LUVs.
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影响因子：
5.6
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通讯作者：
White, SH
影响因子：
2.9
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通讯作者：
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通讯作者：
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作者：
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通讯作者：
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