Department of Chemistry, University of Virginia,Charlottesville, Virginia 22904, United States.
Biochemistry. 2010 Nov 30;49(47):10045-60. doi: 10.1021/bi101148w. Epub 2010 Nov 8.
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 line shapes 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 Aquifex 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 nontraditional 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.
理解膜蛋白在其天然疏水环境中的结构和动力学对于理解这些蛋白质的功能至关重要。电子顺磁共振（EPR）光谱结合定点自旋标记（SDSL）可测量膜蛋白在其天然脂质环境中的结构和动力学；然而，到目前为止，由于对疏水环境中氮氧自由基自旋标记分子内运动的知识有限，所测量的动力学一直是定性的。尽管有几项研究阐明了水溶性蛋白质的 EPR 线宽的结构起源，但在去污剂或脂质中标记氮氧自由基的蛋白质的 EPR 光谱与它们的水溶性对应物有明显的差异，这表明自旋标记在两种环境下的分子运动有很大的不同。为了阐明这些差异，使用 X 射线晶体学、突变分析、氮氧自由基侧链衍生物和光谱模拟研究了来自 Aquifex aeolicus 的亮氨酸转运蛋白 LeuT 的膜暴露α-螺旋位点，以获得氮氧自由基的运动模型。对于每个晶体结构，二硫键连接的自旋标记侧链（R1）的氮氧自由基环被解析，并与蛋白质表面的疏水性残基接触。LeuT 上的 I204 位的自旋标记与最近邻 F208 的邻位氢形成非传统氢键，而 F177 位的自旋标记与相邻螺旋形成的疏水性口袋形成多个范德华接触。这些结果以及突变 i ± 3、4 个残基的光谱效应表明，与水溶性蛋白质表面相比，自旋标记在低介电环境中与局部蛋白质环境具有更高的亲和力。这里呈现的 EPR 光谱模拟表明，自旋标记在保持与蛋白质表面微弱接触的同时，围绕最靠近环的末端键振荡。综合这些结果，为确定膜蛋白上 R1 的运动模型提供了起点，允许在去污剂和脂质的脂肪族环境中定量氮氧自由基的动力学。此外，还提供了 R1 在膜蛋白上的旋转异构体库的初步贡献，这将有助于可靠地模拟 R1 的构象空间，用于脉冲偶极 EPR 和 NMR 顺磁弛豫增强距离测定。
