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首页> 外文期刊>Biochemistry >The Membrane Interface Dictates Different Anchor Roles for “Inner Pair” and “Outer Pair” Tryptophan Indole Rings in Gramicidin A Channels
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The Membrane Interface Dictates Different Anchor Roles for “Inner Pair” and “Outer Pair” Tryptophan Indole Rings in Gramicidin A Channels

机译：膜界面决定了格拉米西汀A通道中“内对”和“外对”色氨酸吲哚环的不同锚定作用

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We investigated the effects of substituting two of the four tryptophans (the “inner pair”nTrp9nand Trp11nor the “outer pair” Trp13nand Trp15n) in gramicidin A (gA) channels. Thenconformational preferences of the doubly substituted gA analogues were assessed using circularndichroism spectroscopy and size-exclusion chromatography, which show that the inner tryptophans 9nand 11 are critical for the gA’s conformational preference in lipid bilayer membranes. [Phen13,15n]gAnlargely retains the single-stranded helical channel structure, whereas [Phen9,11n]gA exists primarily asndouble-stranded conformers.Within this context, then2nH NMR spectra from labeled tryptophans werenused to examine the changes in average indole ring orientations, induced by the Phe substitutions andnby the shift in conformational preference.Using amethod for deuteriumlabeling of already synthesizedngAs, we introduced deuteriumselectively onto positions C2 andC5 of the remaining tryptophan indolenrings in the substituted gA analogues for solid-staten2nH NMR spectroscopy. The (least possible)nchanges in orientation and overall motion of each indole ring were estimated from the experimentalnspectra. Regardless of the mixture of backbone folds, the indole ring orientations observed in thenanalogues are similar to those found previously for gA channels. Both Phe-substituted analogues form single-stranded channels, asnjudged from the formation of heterodimeric channels with the native gA. [Phen13,15n]gA channels have Nanþ currents that are ∼50%nand lifetimes that are ∼80% of those of native gA channels. The double-stranded conformer(s) of [Phen9,11n]gA do not formndetectable channels. The minor single-stranded population of [Phen9,11n]gA forms channels with Nanþ currents that are ∼25% andnsingle-channel lifetimes that are∼300% of those of native gA channels.Our results suggest that Trp9nand Trp11n, when “reaching” fornthe interface, tend to drive bothmonomer folding (to “open” a channel) and dimer dissociation (to “close” a channel). Furthermore,nthe dipoles of Trp9nand Trp11nare relatively more important for the single-channel conductance than are the dipoles of Trp13nandnTrp15n.
机译：我们研究了在短杆菌肽A（gA）通道中替代四个色氨酸中的两个（“内部对” nTrp9n和Trp11或“外部对” Trp13n和Trp15n）的作用。然后使用圆二色光谱和尺寸排阻色谱法评估了双取代的gA类似物的构象偏好，结果表明内部色氨酸9n和11对于脂质双层膜中gA的构象偏好至关重要。 [Phen13,15n] g基本上保留了单链螺旋通道结构，而[Phen9,11n] gA主要以双链构象形式存在。在这种情况下，随后用标记的色氨酸的2nH NMR光谱检查了吲哚环平均取向的变化，利用Phe取代引起的构象偏倚诱导，并通过构象偏好的转移来诱导氘的形成每个吲哚环的取向和整体运动的（最不可能的）变化是根据实验光谱估计的。不管骨架折叠的混合情况如何，在类似物中观察到的吲哚环取向与以前在gA通道中发现的相似。两种Phe取代的类似物均形成单链通道，这是由与天然gA形成的异二聚体通道所断定的。 [Phen13,15n] gA通道的Nanþ电流约为自然gA通道的〜50％n，寿命约为〜80％。 [Phen9,11n] gA的双链构象体不形成可检测的通道。较小的[Phen9,11n] gA单链群体形成的通道具有约25％的Nanþ电流和约为天然gA通道的约300％的单通道寿命。我们的结果表明，“到达”时，Trp9n和Trp11n在界面之前，趋向于既驱动单体折叠（“打开”通道）又驱动二聚体解离（“关闭”通道）。此外，对于单通道电导，Trp9n和Trp11的偶极比Trp13nandnTrp15n的偶极更为重要。

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