SPIN LABELED GRAMICIDIN A: CHANNEL FORMATION AND DISSOCIATION

旋转标记的短杆菌肽 A：通道形成和解离

• 批准号: 7723897
• 负责人: BORIS G DZIKOVSKI
• 金额: $ 0.48万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723897
• 关键词: Agreement; Back; Biological Models; Computer Retrieval of Information on Scientific Projects Database; Depth; Dimyristoylphosphatidylcholine; Disruption; Dissociation; Environment; Funding; Gel; Gramicidin; Grant; Head; Heating; Helix (Snails); High temperature of physical object; Institution; Length; Lipids; Membrane; Membrane Lipids; Membrane Proteins; Methods; Molecular Conformation; Phase; Phase Transition; Pliability; Research; Research Personnel; Resources; Source; Spin Labels; Surface; Thick; United States National Institutes of Health; Variant; Vertebral column; Vesicle; dimer; ear helix; gramicidin A; simulation

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 合成了自旋标记的短杆菌肽 A (GAsl)，并通过 ESR、高场 ESR 和 DQC-ESR 在不同的脂质膜中进行研究，通过 ISDU 进行显微排列并在囊泡中进行研究。这是一个用于开发膜蛋白方法的优秀模型系统。 DQC 清楚地表明 DMPC 膜中存在成对，以及由不饱和脂质或比 DMPC 短的饱和脂质构成的膜。成对的自旋距离（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。尽管这种差异可能是由于硝基氧系链的灵活性造成的，但我们不能排除通道二聚体的主链长度也可能是脂质依赖性的。 ESR 谱分析显示头对头二聚体深度嵌入和硝基氧基团的倾斜。 在 DPPC 和 DSPC GAsl 的 L-β 相中，GAsl 呈现出不同的、显然是双螺旋的构象。在这种情况下，硝基氧部分显示出良好的 Z 排序，并且位于靠近膜表面的位置，DQC 检测到两个以上分子的聚集迹象。凝胶相中的DPPC和DSPC在二聚体长度和双层厚度之间具有较大的疏水失配，因此它们显然不利于通道形成。 DPPC 双螺旋中的 L-β 和 P-β 相变以上转变为通道。由于硝基氧部分的倾斜及其环境极性的降低，通道的形成表现为 Z 排序的破坏。经过大量频谱模拟后，可以从 9GHz 频谱得出这一结论，即使通过检查，在 170GHz 下也非常清楚。通过在对齐的 DPPC 膜中执行冷却/加热循环，光谱强度从 Z 区域移动到 XY 区域并返回。我们将循环中相当大的滞后归因于通道形式的缓慢解离。滞后现象使我们能够锁定高温构象并通过 DQC-ESR 观察 P-β 相通道的形成。