Hustedt E J, Beth A H
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232-0615, USA.
Biochemistry. 1996 May 28;35(21):6944-54. doi: 10.1021/bi9601518.
The orientation of the nitroxide moiety of an isotopically substituted spin-labeled derivative of dihydrostilbenedisulfonate ([15N,2H13]-SL-H2DADS-maleimide) covalently coupled at the extracellular stilbenedisulfonate binding site of the human erythrocyte anion exchange protein, band 3, has been determined relative to the membrane normal axis of intact cells. The X-band linear electron paramagnetic resonance (EPR) spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled band 3 in intact erythrocytes oriented by flow through an EPR flat cell have been obtained for two orthogonal orientations of the sample in the DC magnetic field. Two different methods of analysis have provided very similar values for the angles alpha 1 and beta 1 which uniquely define the orientation of the nitroxide axis frame relative to the membrane normal axis. In the first approach, a variable fraction of the cells, f, were taken to be biconcave disks perfectly oriented relative to the flat cell surface with the remainder, 1-f, isotropically oriented. Simultaneous nonlinear least squares analysis of the spectra obtained at the two sample orientations yielded best fit values of f = 0.60, alpha 1 = 58 degrees, and beta 1 = 36 degrees. In the second approach, the EPR spectra of flow-oriented intact erythrocytes labeled with the fatty acid spin-label, [15N,2H12]-5-nitroxyl stearate, have been obtained at the two sample orientations. These two spectra have been used to determine a model-independent distribution of membrane normal orientations in the sample. Using this experimentally determined membrane normal orientation distribution, the EPR spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled erythrocytes were then reanalyzed to obtain a second determination of the nitroxide orientation, alpha 1 = 61 degrees and beta 1 = 37 degrees. The orientation of the nitroxide with respect to the membrane normal axis determined in the present study is nearly identical to the orientation of the nitroxide with respect to the uniaxial rotational diffusion axis, alpha = 66 degrees and beta = 34 degrees, as determined from saturation transfer EPR (ST-EPR) studies [Hustedt, E. H., & Beth, A. H. (1995) Biophys. J. 69, 1409-1423]. This result supports the conclusion that the motion observed using ST-EPR spectroscopy is, in fact, the uniaxial rotational diffusion of band 3 about the membrane normal.
二氢茋二磺酸盐的同位素取代自旋标记衍生物（[15N,2H13]-SL-H2DADS-马来酰亚胺）与人红细胞阴离子交换蛋白带3的细胞外茋二磺酸盐结合位点共价偶联，其氮氧化物部分相对于完整细胞的膜法线轴的取向已被确定。通过在EPR扁平池中流动使完整红细胞中的[15N,2H13]-SL-H2DADS-马来酰亚胺标记的带3取向，针对样品在直流磁场中的两个正交取向获得了X波段线性电子顺磁共振（EPR）光谱。两种不同的分析方法为角度α1和β1提供了非常相似的值，这两个角度唯一地定义了氮氧化物轴系相对于膜法线轴的取向。在第一种方法中，将可变比例的细胞f视为相对于扁平细胞表面完美取向的双凹圆盘，其余部分1-f为各向同性取向。对在两个样品取向获得的光谱进行同时非线性最小二乘分析，得到f = 0.60、α1 = 58°和β1 = 36°的最佳拟合值。在第二种方法中，在两个样品取向获得了用脂肪酸自旋标记物[15N,2H12]-5-硝基硬脂酸标记的流动取向完整红细胞的EPR光谱。这两个光谱已用于确定样品中膜法线取向的与模型无关的分布。利用这个实验确定的膜法线取向分布，然后对[15N,2H13]-SL-H2DADS-马来酰亚胺标记的红细胞的EPR光谱进行重新分析，以获得氮氧化物取向的第二次测定值，α1 = 61°和β1 = 37°。本研究中确定的氮氧化物相对于膜法线轴的取向与通过饱和转移EPR（ST-EPR）研究[Hustedt, E. H., & Beth, A. H. (1995) Biophys. J. 69, 1409 - 1423]确定的氮氧化物相对于单轴旋转扩散轴的取向α = 66°和β = 34°几乎相同。这一结果支持了这样的结论，即使用ST-EPR光谱观察到的运动实际上是带3围绕膜法线的单轴旋转扩散。
