THE AGGREGATION STATE OF SPIN-LABELED MELITTIN IN SOLUTION AND BOUND TO PHOSPHOLIPID-MEMBRANES - EVIDENCE THAT MEMBRANE-BOUND MELITTIN IS MONOMERIC
THE AGGREGATION STATE OF SPIN-LABELED MELITTIN IN SOLUTION AND BOUND TO PHOSPHOLIPID-MEMBRANES - EVIDENCE THAT MEMBRANE-BOUND MELITTIN IS MONOMERIC
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DOI：
10.1002/prot.340030404
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发表时间：
1988-01-01
影响因子：
2.9
通讯作者：
HUBBELL, WL
中科院分区：
文献类型：
--
作者：
ALTENBACH, C;HUBBELL, WL
蜂毒蛋白的自旋标记衍生物是通过蛋白质的四个氨基中的平均一个与琥珀酰亚胺基-2，2，5，5-四甲基-3-吡咯啉-1-氧基-3-羧酸酯反应而获得的。然后用反相高效液相色谱法一次性分离所有16种统计学上可能的反应产物，每种蛋白质具有0、1、2、3或4个自旋标记。在胰蛋白酶消化和二极管阵列检测的帮助下，可以分配所有16个洗脱级分的一级结构。纯化每个蛋白质仅具有一个自旋标记的所有级分用于电子顺磁共振测量。标记位点覆盖蛋白质的不同区域：一个在N末端，一个在赖氨酸-7，两个分别在赖氨酸-21和赖氨酸-23的C末端附近。这套专门标记的蜂毒肽被用来研究蜂毒肽在水溶液中的结构和动力学，当绑定到中性或带负电荷的膜。在水溶液中，在盐诱导的从无规卷曲单体到主要为α-螺旋的转变期间，观察到旋转相关时间的减少和自旋-自旋相互作用的出现。螺旋四聚体在低或高离子强度下膜与磷脂双层的结合仅反映在流动性的进一步降低中。在膜结合状态下没有任何自旋相互作用表明蜂毒肽在这些条件下是单体的。所有衍生物都能够检测到这些结构变化，但蜂毒肽标记的N-末端氨基是特别有价值的。由于假定的分子内氢键，这种标记直接反映了整个蛋白质或四聚体的运动。用草酸铬的扩大实验表明，所有标记的网站至少部分暴露于水相时蜂毒肽结合到膜。这表明，α-螺旋蜂毒素单体以其轴平行于膜表面的方式结合到膜上。
Spin-labeled derivatives of the bee venom protein, melittin, were obtained by reacting on the average one of the four amino groups of the protein with succinimidyl-2,2,5,5-tetramethyl-3-pyroline-1-oxyl-3-carboxylate. All 16 statistically possible reaction products with 0, 1, 2, 3, or 4 spin labeles per protein were then separated in a single pass with reversed phase high performance liquid chromatography. With the help of trypsin digestion and diode array detection it was possible to assign the primary structure of all 16 eluting fractions. All fractions with only one spin label per protein were purified for electron paramagnetic resonance measurements. The labeling sites cover different regions of the protein: one is at the N-terminus, one at lysine-7, and two are near the C-terminus at lysine-21 and lysine-23, respectively. This set of specifically labeled mellitins was used to study the structure and dynamics of melittin in aqueous solutions and when bound to neutral or negatively charged membranes. In aqueous solution a reduction in rotational correlation time and appearance of spin-spin interaction was observed during salt-induced transition from a random coil monomer to a mostly .alpha.-helical tetramer. Membrane binding to phospholipid bilayers in low or high ionic strength was reflected only in a further decrease in mobility. The absence of any spin interaction in the membrane-bound state suggests that melittin is monomeric under these conditions. All derivatives were able to detect these structural changes, but melittin labeled at the N-terminal amino group was especially valuable. Because of postulated intramolecular hydrogen bonding, this label reflects directly the motion of the entire protein or tetramer. Broadening experiments with chromium oxalate show that all labeled sites are at least partially exposed to the aqueous phase when mellitin is bound to membranes. This suggests that an .alpha.-helical melittin monomer binds to membranes with its axis parallel to the membrane surface.