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首页> 外文期刊>Biochemistry >Membrane Orientation and Position of the C2 Domain from cPLA2 by Site-Directed Spin Labeling.
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Membrane Orientation and Position of the C2 Domain from cPLA2 by Site-Directed Spin Labeling.

机译：通过位置定向自旋标记从cPLA2获得的C2域的膜方向和位置。

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摘要

The C2 domain is a ubiquitous Ca(2+)-binding motif that triggers the membrane docking of many key signaling proteins during intracellular Ca(2+) signals. Site-directed spin labeling was carried out on the C2 domain of cytosolic phospholipase A(2) in order to determine the depth of penetration and orientation of the domain at the membrane interface. Membrane depth parameters, Phi, were obtained by EPR spectroscopy for a series of selectively spin-labeled C2 domain cysteine mutants, and for spin-labeled lipids and spin-labeled bacteriorhodopsin cysteine mutants. Values of Phi were combined with several other constraints, including the solution NMR structure, to generate a model for the position of the C2 domain at the membrane interface. This modeling yielded an empirical expression for Phi, which for the first time defines its behavior from the bulk aqueous phase to the center of the lipid bilayer. In this model, the backbones of both the first and third Ca(2+)-binding loops are inserted approximately 10 A into the bilayer, with residues inserted as deep as 15 A. The backbone of the second Ca(2+)-binding loop is positioned near the lipid phosphate, and the two beta-sheets of the C2 domain are oriented so that the individual strands make angles of 30-45 degrees with respect to the bilayer surface. Upon membrane docking, spin labels in the Ca(2+)-binding loops exhibit decreases in local motion, suggesting either changes in tertiary contacts due to protein conformational changes and/or interactions with lipid.
机译：C2域是一个普遍存在的Ca（2+）结合基序，在细胞内Ca（2+）信号期间触发许多关键信号蛋白的膜对接。在细胞溶质磷脂酶A（2）的C2域上进行了定点自旋标记，以便确定该域在膜界面处的渗透深度和方向。通过EPR光谱法获得了一系列深度自旋标记的C2域半胱氨酸突变体，自旋标记的脂质和自旋标记的细菌视紫红质半胱氨酸突变体的膜深度参数Phi。将Phi值与其他几个约束条件（包括溶液NMR结构）组合起来，以生成C2域在膜界面位置的模型。该模型产生了一个针对Phi的经验表达式，该表达式首次定义了从大体积水相到脂质双层中心的行为。在此模型中，第一个和第三个Ca（2+）结合环的骨架被插入到双层中大约10 A，残基插入深度为15A。第二个Ca（2+）结合的骨架环位于脂质磷酸酯附近，并且C2结构域的两个β-折叠定向，使得各个链相对于双层表面成30-45度的角度。在膜对接后，在Ca（2+）结合环中的自旋标记显示局部运动的减少，表明由于蛋白质构象变化和/或与脂质的相互作用而引起的第三级接触的变化。

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