A COLLISION GRADIENT-METHOD TO DETERMINE THE IMMERSION DEPTH OF NITROXIDES IN LIPID BILAYERS - APPLICATION TO SPIN-LABELED MUTANTS OF BACTERIORHODOPSIN
A COLLISION GRADIENT-METHOD TO DETERMINE THE IMMERSION DEPTH OF NITROXIDES IN LIPID BILAYERS - APPLICATION TO SPIN-LABELED MUTANTS OF BACTERIORHODOPSIN
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DOI：
10.1073/pnas.91.5.1667
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发表时间：
1994-03-01
影响因子：
11.1
通讯作者：
HUBBELL, WL
中科院分区：
文献类型：
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作者：
ALTENBACH, C;GREENHALGH, DA;HUBBELL, WL
细菌视紫红质的10个突变体，每个突变体含有一个半胱氨酸残基，沿沿着螺旋D有规律地间隔开，并面向脂质双层，衍生与氮氧自旋标记。氮氧化合物与非极性氧的碰撞速率随膜/溶液界面距离的增加而增加。与极性金属离子络合物的碰撞率在相同的距离上下降。虽然碰撞率取决于由局部蛋白质结构和膜中的深度所施加的空间约束，但氧与极性金属离子络合物的碰撞率的比率与蛋白质的结构特征无关。比率的对数是膜内深度的线性函数。用自旋标记的磷脂校准这个比率参数允许定位各个氮氧自由基，因此细菌视紫红质分子，相对于双层的磷酸基团的平面。残基之间的间距与α-螺旋的螺距一致。这些结果为确定氮氧化合物在双层膜中的浸没深度提供了一种通用策略。
Ten mutants of bacteriorhodopsin, each containing a single cysteine residue regularly spaced along helix D and facing the lipid bilayer, were derivatized with a nitroxide spin label. Collision rates of the nitroxide with apolar oxygen increased with distance from the membrane/solution interface. Collision rates with polar metal ion complexes decreased over the same distance. Although the collision rates depend on steric constraints imposed by the local protein structure and on the depth in the membrane, the ratio of the collision rate of oxygen to those of a polar metal ion complex is independent of structural features of the protein. The logarithm of the ratio is a linear function of depth within the membrane. Calibration of this ratio parameter with spin-labeled phospholipids allows localization of the individual nitroxides, and hence the bacteriorhodopsin molecule, relative to the plane of the phosphate groups of the bilayer. The spacing between residues is consistent with the pitch of an alpha-helix. These results provide a general strategy for determining the immersion depth of nitroxides in bilayers.