Structural origins of nitroxide side chain dynamics on membrane protein α-helical sites.
Structural origins of nitroxide side chain dynamics on membrane protein α-helical sites.
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DOI：
10.1021/bi101148w
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发表时间：
2010-11-30
期刊：
影响因子：
2.9
通讯作者：
Columbus, Linda
中科院分区：
文献类型：
--
作者：
Kroncke, Brett M.;Horanyi, Peter S.;Columbus, Linda
了解膜蛋白在其天然疏水环境中的结构和动力学对于了解这些蛋白质如何发挥功能非常重要。EPR光谱结合定点自旋标记（SDSL）可以测量膜蛋白在其天然脂质环境中的动力学和结构;然而，到目前为止，由于对氮氧自旋标记在疏水环境中的分子内运动的了解有限，所测量的动力学一直是定性的。虽然有几项研究已经阐明了水溶性蛋白质的EPR线型的结构起源，EPR光谱的氮氧化物自旋标记的蛋白质在洗涤剂或脂质具有特征的差异，从他们的水溶性对应物表明显着的差异，在两种环境之间的自旋标记的基本分子运动。为了阐明这些差异，膜暴露的亮氨酸转运蛋白，LeuT，从A。aeolicus，进行了研究，使用X射线晶体学，突变分析，氮氧侧链衍生物，和光谱模拟，以获得一个运动模型的氮氧。对于每个晶体结构，二硫键连接的自旋标记侧链（R1）的氮氧环被解析，并与蛋白质表面上的疏水残基接触。LeuT上位点I204处的自旋标记与其最近邻F208上的邻位氢形成非传统氢键，而位点F177处的自旋标记与由相邻螺旋形成的疏水口袋形成多个货车德瓦耳斯接触。这些结果与突变i ± 3，4残基的光谱效应相结合，表明自旋标记物在低介电介质中对其局部蛋白质环境的亲和力比在水溶性蛋白质表面上更大。这里提出的EPR谱的模拟表明，自旋标签振荡的最接近的环的末端键，同时保持与蛋白质表面的弱接触。结合起来，结果提供了一个起点，用于确定一个运动模型R1膜蛋白允许定量的氮氧动力学在脂肪族环境中的洗涤剂和脂质。此外，最初的贡献R1膜蛋白的旋转异构体库提供，这将有助于可靠地建模脉冲偶极EPR和NMR顺磁弛豫增强距离测定的R1构象空间。
Understanding the structure and dynamics of membrane proteins in their native, hydrophobic environment is important to understanding how these proteins function. EPR spectroscopy in combination with site directed spin labeling (SDSL) can measure dynamics and structure of membrane proteins in their native lipid environment; however, until now the dynamics measured have been qualitative due to limited knowledge of the nitroxide spin label’s intramolecular motion in the hydrophobic environment. Although several studies have elucidated the structural origins of EPR lineshapes of water-soluble proteins, EPR spectra of nitroxide spin labeled proteins in detergents or lipids have characteristic differences from their water-soluble counterparts suggesting significant differences in the underlying molecular motion of the spin label between the two environments. To elucidate these differences, membrane exposed α-helical sites of the leucine transporter, LeuT, from A. aeolicus, were investigated using X-ray crystallography, mutational analysis, nitroxide side chain derivatives, and spectral simulations in order to obtain a motional model of the nitroxide. For each crystal structure, the nitroxide ring of a disulfide-linked spin label side chain (R1) is resolved and makes contacts with hydrophobic residues on the protein surface. The spin label at site I204 on LeuT makes a non-traditional hydrogen bond with the ortho hydrogen on its nearest neighbor F208, whereas the spin label at site F177 makes multiple van der Waals contacts with a hydrophobic pocket formed with an adjacent helix. These results coupled with the spectral effect of mutating the i ± 3, 4 residues suggest that the spin label has a greater affinity for its local protein environment in the low dielectric than on a water-soluble protein surface. The simulations of the EPR spectra presented here suggest the spin label oscillates about the terminal bond nearest the ring while maintaining weak contact with the protein surface. Combined, the results provide a starting point for determining a motional model for R1 on membrane proteins allowing quantification of nitroxide dynamics in the aliphatic environment of detergent and lipids. In addition, initial contributions to a rotamer library of R1 on membrane proteins are provided, which will assist in reliably modeling the R1 conformational space for pulsed dipolar EPR and NMR paramagnetic relaxation enhancement distance determination.
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影响因子：
3.4
作者：
Chamberlain, AK;Bowie, JU
通讯作者：
Bowie, JU
影响因子：
2.9
作者：
Altenbach, C;Yang, K;Hubbell, WL
通讯作者：
Hubbell, WL
影响因子：
15
作者：
Gu, YL;Kar, T;Scheiner, S
通讯作者：
Scheiner, S
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
8
作者：
Guo, Zhefeng;Cascio, Duilio;Hubbell, Wayne L.
通讯作者：
Hubbell, Wayne L.