Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, United States.
Biochemistry. 2011 Jun 7;50(22):4855-66. doi: 10.1021/bi200136e. Epub 2011 May 13.
We investigated the effects of substituting two of the four tryptophans (the "inner pair" Trp(9) and Trp(11) or the "outer pair" Trp(13) and Trp(15)) in gramicidin A (gA) channels. The conformational preferences of the doubly substituted gA analogues were assessed using circular dichroism spectroscopy and size-exclusion chromatography, which show that the inner tryptophans 9 and 11 are critical for the gA's conformational preference in lipid bilayer membranes. [Phe(13,15)]gA largely retains the single-stranded helical channel structure, whereas [Phe(9,11)]gA exists primarily as double-stranded conformers. Within this context, the (2)H NMR spectra from labeled tryptophans were used to examine the changes in average indole ring orientations, induced by the Phe substitutions and by the shift in conformational preference. Using a method for deuterium labeling of already synthesized gAs, we introduced deuterium selectively onto positions C2 and C5 of the remaining tryptophan indole rings in the substituted gA analogues for solid-state (2)H NMR spectroscopy. The (least possible) changes in orientation and overall motion of each indole ring were estimated from the experimental spectra. Regardless of the mixture of backbone folds, the indole ring orientations observed in the analogues are similar to those found previously for gA channels. Both Phe-substituted analogues form single-stranded channels, as judged from the formation of heterodimeric channels with the native gA. [Phe(13,15)]gA channels have Na(+) currents that are ~50% and lifetimes that are ~80% of those of native gA channels. The double-stranded conformer(s) of [Phe(9,11)]gA do not form detectable channels. The minor single-stranded population of [Phe(9,11)]gA forms channels with Na(+) currents that are ~25% and single-channel lifetimes that are ~300% of those of native gA channels. Our results suggest that Trp(9) and Trp(11), when "reaching" for the interface, tend to drive both monomer folding (to "open" a channel) and dimer dissociation (to "close" a channel). Furthermore, the dipoles of Trp(9) and Trp(11) are relatively more important for the single-channel conductance than are the dipoles of Trp(13) and Trp(15).
我们研究了在革兰菌素 A (gA) 通道中替换四个色氨酸中的两个（“内对”色氨酸 9 和 11 或“外对”色氨酸 13 和 15）的效果。使用圆二色性光谱和尺寸排阻色谱法评估了双取代 gA 类似物的构象偏好，结果表明内色氨酸 9 和 11 对于 gA 在脂质双层膜中的构象偏好至关重要。[Phe(13,15)]gA 主要保留单链螺旋通道结构，而 [Phe(9,11)]gA 主要存在双链构象。在这种情况下，通过对标记色氨酸的 (2)H NMR 光谱进行研究，我们检查了由 Phe 取代和构象偏好改变引起的平均吲哚环取向的变化。使用一种已合成的 gA 的氘标记方法，我们在取代的 gA 类似物中的剩余色氨酸吲哚环的 C2 和 C5 位置上选择性地引入氘，用于固态 (2)H NMR 光谱。从实验光谱中估计了每个吲哚环的取向和整体运动的 (最小可能) 变化。无论骨架折叠的混合物如何，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 取代的类似物都形成单链通道，这可以从与天然 gA 形成异二聚体通道来判断。[Phe(13,15)]gA 通道的 Na(+)电流约为天然 gA 通道的 50%，寿命约为 80%。[Phe(9,11)]gA 的双链构象(s) 不能形成可检测的通道。[Phe(9,11)]gA 的少量单链种群形成的通道的 Na(+)电流约为天然 gA 通道的 25%，单通道寿命约为 300%。我们的结果表明，当 Trp(9)和 Trp(11)“到达”界面时，它们倾向于同时驱动单体折叠（“打开”通道）和二聚体解离（“关闭”通道）。此外，Trp(9)和 Trp(11)的偶极子对于单通道电导比 Trp(13)和 Trp(15)的偶极子更重要。
