A ruler for determining the position of proteins in membranes
A ruler for determining the position of proteins in membranes
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DOI：
10.1021/ja042782s
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发表时间：
2005-05-04
影响因子：
15
通讯作者：
Robinson, BH
中科院分区：
文献类型：
--
作者：
Nielsen, RD;Che, KP;Robinson, BH
氧的扩散速率和氧的溶解度随进入生物膜内部的深度而变化。这两个梯度的乘积产生一个单一的梯度，即渗透梯度，它是离膜中心距离的平滑连续函数。利用电子顺磁共振和自旋探针法，氧的弛豫梯度是生理条件下膜内可直接测量的量，与透性梯度成正比。得到的梯度为确定膜结合蛋白环区或跨膜蛋白暴露在膜上的残基的膜深度提供了校准标尺。我们已经确定了氧在两性离子和阴离子磷脂膜上的弛豫梯度，通过将单个氮氧化物探针连接到跨膜α -螺旋多肽的特定残基上。肽尺用于测定胞质磷脂酶A C2结构域钙结合环的渗透深度(2)。这种膜结合蛋白穿透膜的选定残基的位置，用这种尺子确定，与以前使用更复杂的方法确定的位置相比有利。弛豫梯度限制了膜依赖氧浓度和氧扩散梯度的可能值。据估计，氧在膜中的平均扩散系数至少比在水中的扩散系数小2倍。
Both the oxygen diffusion rate and the oxygen solubility vary with depth into the interior of biological membranes. The product of these two gradients generates a single gradient, a permeability gradient, which is a smooth continuous function of the distance from the center of the membrane. Using electron paramagnetic resonance and the spin-probe method, the relaxation gradient of oxygen, which is directly proportional to the permeability gradient, is the quantity that can be directly measured in membranes under physiological conditions. The gradient obtained provides a calibrated ruler for determining the membrane depth of residues either from loop regions of membrane-binding proteins or from the membrane-exposed residues of transmembrane proteins. We have determined the relaxation gradient of oxygen in zwitterionic and anionic phospholipid membranes by attaching a single nitroxide probe to a transmembrane alpha-helical polypeptide at specific residues. The peptide ruler was used to determine the depth of penetration of the calcium-binding loops of the C2 domain of cytosolic phospholipase A(2). The positions of selected residues of this membrane-binding protein that penetrate into the membrane, determined using this ruler, compared favorably with previous determinations using more complex methods. The relaxation gradient constrains the possible values of the membrane-dependent oxygen concentration and the oxygen diffusion gradients. The average oxygen diffusion coefficient is estimated to be at least 2-fold smaller in the membrane than that in water.