Vogel Alexander, Scheidt Holger A, Huster Daniel
Junior Research Group Solid-state NMR Studies of the Structure of Membrane-associated Proteins, Biotechnological-Biomedical Center, Institute of Medical Physics and Biophysics, University of Leipzig, D-04103 Leipzig, Germany.
Biophys J. 2003 Sep;85(3):1691-701. doi: 10.1016/S0006-3495(03)74599-8.
The distribution of the lipid-attached doxyl electron paramagnetic resonance (EPR) spin label in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes has been studied by (1)H and (13)C magic angle spinning nuclear magnetic resonance relaxation measurements. The doxyl spin label was covalently attached to the 5th, 10th, and 16th carbons of the sn-2 stearic acid chain of a 1-palmitoyl-2-stearoyl-(5/10/16-doxyl)-sn-glycero-3-phosphocholine analog. Due to the unpaired electron of the spin label, (1)H and (13)C lipid relaxation rates are enhanced by paramagnetic relaxation. For all lipid segments the influence of paramagnetic relaxation is observed even at low probe concentrations. Paramagnetic relaxation rates provide a measure for the interaction strength between lipid segments and the doxyl group. Plotted along the membrane director a transverse distribution profile of the EPR probe is obtained. The chain-attached spin labels are broadly distributed in the membrane with a maximum at the approximate chain position of the probe. Both (1)H and (13)C relaxation measurements show these broad distributions of the doxyl group in the membrane indicating that (1)H spin diffusion does not influence the relaxation measurements. The broad distributions of the EPR label result from the high degree of mobility and structural heterogeneity in liquid-crystalline membranes. Knowing the distribution profiles of the EPR probes, their influence on relaxation behavior of membrane inserted peptide and protein segments can be studied by (13)C magic angle spinning nuclear magnetic resonance. As an example, the location of Ala residues positioned at three sites of the transmembrane WALP-16 peptide was investigated. All three doxyl-labeled phospholipid analogs induce paramagnetic relaxation of the respective Ala site. However, for well ordered secondary structures the strongest relaxation enhancement is observed for that doxyl group in the closest proximity to the respective Ala. Thus, this approach allows study of membrane insertion of protein segments with respect to the high molecular mobility in liquid-crystalline membranes.
通过（1）H和（13）C魔角旋转核磁共振弛豫测量，研究了脂质连接的多羟基电子顺磁共振（EPR）自旋标记物在1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱膜中的分布。多羟基自旋标记物共价连接到1-棕榈酰-2-硬脂酰-（5/10/16-多羟基）-sn-甘油-3-磷酸胆碱类似物的sn-2硬脂酸链的第5、10和16个碳原子上。由于自旋标记物的未成对电子，（1）H和（13）C脂质弛豫率因顺磁弛豫而增强。对于所有脂质片段，即使在低探针浓度下也能观察到顺磁弛豫的影响。顺磁弛豫率提供了脂质片段与多羟基基团之间相互作用强度的一种度量。沿膜取向绘制可得到EPR探针的横向分布轮廓。链连接的自旋标记物在膜中广泛分布，在探针的近似链位置处有一个最大值。（1）H和（13）C弛豫测量均显示多羟基基团在膜中的这些广泛分布，表明（1）H自旋扩散不影响弛豫测量。EPR标记物的广泛分布源于液晶膜中的高度流动性和结构异质性。了解EPR探针的分布轮廓后，可通过（13）C魔角旋转核磁共振研究它们对插入膜中的肽和蛋白质片段弛豫行为的影响。例如，研究了位于跨膜WALP-16肽三个位点的丙氨酸残基的位置。所有三种多羟基标记的磷脂类似物均诱导各自丙氨酸位点的顺磁弛豫。然而，对于有序的二级结构，在最接近各自丙氨酸的那个多羟基基团处观察到最强的弛豫增强。因此，这种方法允许研究蛋白质片段在液晶膜中相对于高分子流动性的膜插入情况。
