SPIN LABELED GRAMICIDIN A: CHANNEL FORMATION AND DISSOCIATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 合成了自旋标记短杆菌肽A（GAsl），并用ESR、高场ESR和DQC-ESR研究了GAsl在不同脂膜、ISDU显微排列和囊泡中的作用。这是一个很好的模型系统，用于开发我们的膜蛋白方法。DQC清楚地表明DMPC膜中存在配对，以及膜中由不饱和脂质或比DMPC短的饱和脂质组成。成对的自旋间距离（DMPC为30.9 <$）与我们对头对头二聚体的估计非常一致，根据脂质的不同，变化很小。虽然这是可能的，差异可能会导致在氮氧系链的灵活性，我们不能排除的通道二聚体的主链长度也可能是脂质依赖性。ESR谱分析表明，头对头二聚体的深嵌入和氮氧基团的倾斜。 在DPPC和DSPC的L-β相中，GAsl呈现不同的明显双螺旋构象。在这种情况下，氮氧部分显示出良好的Z-排序，并位于膜表面附近，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具有大的疏水性失配，因此它们显然不利于通道形成。以上DPPC双螺旋中的L-β和P-β相变转变为通道。通道的形成表现为由于氮氧部分的倾斜和其环境的极性降低而导致的Z-有序的破坏。这一结论可以在广泛的频谱模拟后从9 GHz频谱中确定，即使通过检查也可以在170 GHz处非常清楚。通过在对齐的DPPC膜中进行冷却/加热循环，光谱强度从Z区域移动到XY区域并返回。我们将循环中的相当大的滞后归因于通道形式的缓慢解离。滞后允许我们锁定在高温构象，并通过DQC-ESR观察在P-β相中的通道形成。

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.