SPIN LABELED GRAMICIDIN A: CHANNEL FORMATION AND DISSOCIATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 合成了自旋标记的甘草素A(GASL)，并用ESR、高场ESR和DQC-ESR研究了GASL在不同脂膜、ISDU显微镜下和囊泡中的分布。这是一个很好的模型系统，可以用来开发我们的膜蛋白方法。DQC清楚地表明DMPC膜中存在对，以及由不饱和脂类或比DMPC短的饱和脂类组成的膜。成对的自旋间距(DMPC为30.9°)与我们对头对头二聚体的估计很好地一致，根据脂质的不同，变化很小。尽管这种差异可能是由于氮氧化物纽带的灵活性造成的，但我们不能排除通道二聚体的主干长度也可能与脂类有关。ESR谱分析表明，二聚体嵌入较深，氮氧基发生倾斜。在DPPC和DSPC的L-β阶段，GASL呈现出不同的、明显的双螺旋构象。在这种情况下，氮氧化物部分表现出良好的Z有序性，并且位于膜表面附近，DQC检测到两个以上分子聚集的迹象。凝胶相中的DPPC和DSPC在二聚体长度和双层厚度之间都有较大的疏水失配，因此它们显然不利于通道的形成。以上L-β和P-β相变在DPPC中双螺旋转化为通道。通道的形成表现为由于氮氧化物部分的倾斜和环境极性的降低而破坏了Z有序性。这一结论可以通过广泛的光谱模拟从9 GHz频谱中确定，即使通过检验，这一结论在170 GHz也是非常清楚的。通过在定向的DPPC膜中执行冷却/加热循环，光谱强度从Z区转移到XY区并返回。我们将周期中相当大的滞后归因于通道形式的缓慢解离。该滞后效应使我们能够锁定高温构象，并通过DQC-ESR观察P-β相的通道形成。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Spin labeled gramicidin A (GAsl) was synthesized and studied by ESR, high-field ESR and DQC-ESR in different lipid membranes, microscopically aligned by ISDU and in vesicles. This is an excellent model system for developing our methods for membrane proteins. DQC clearly indicates the presence of pairs in DMPC membranes, as well as membranes build of unsaturated lipids or saturated lipids shorter than DMPC. The interspin distance in pairs (30.9 ¿ for DMPC) is in good agreement with our estimates for head-to-head dimers, with small variations, depending on the lipid. Though it is possible that the difference may result from flexibility in the nitroxide tethers, we cannot rule out that the backbone length of the channel dimer may also be lipid-dependent. The analysis of ESR spectra shows for the head-to-head dimer deep embedding and a tilt of the nitroxide group. In the L-beta phase of DPPC and DSPC GAsl takes on a different, apparently double helical, conformation. In this case the nitroxide moiety shows good Z-ordering and is located close to the membrane surface, DQC detects signs of aggregation of more than two molecules. Both DPPC and DSPC in the gel phase have a large hydrophobic mismatch between the dimer length and bilayer thickness, so they apparently do not favor channel formation. Above the L-beta and P-beta phase transition in DPPC double helices transform to channels. The channel formation manifests itself as a disruption of Z-ordering due to the tilt of the nitroxide moiety and a decrease in the polarity of its environment. This conclusion, which could be determined from 9GHz spectra after extensive spectral simulations is very clear at 170GHz even by inspection. The spectral intensity shifts from the Z-region to the XY-region and back by performing a cooling/heating cycle in the aligned DPPC membrane. We attribute the considerable hysteresis in the cycle to a slow dissociation of the channel form. The hysteresis allows us to lock-in the high temperature conformation and to observe the channel formation in the P-beta phase by DQC-ESR.