95 GHZ 2D-ELDOR OF SPIN-LABELED PEPTIDES IN MEMBRANES

膜中自旋标记肽的 95 GHZ 2D-ELDOR

• 批准号: 8363964
• 负责人: KEITH A EARLE
• 金额: $ 1.02万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363964
• 关键词: Discrimination; Frequencies; Funding; Grant; Ion Channel; Lipids; Membrane; Membrane Lipids; Membrane Proteins; Microtome - medical device; Modeling; Molecular; National Center for Research Resources; Pattern; Peptides; Physiologic pulse; Principal Investigator; Property; Relaxation; Research; Research Infrastructure; Resolution; Resources; Sampling; Source; Spin Labels; Structure; Techniques; Technology; Temperature; Time; United States National Institutes of Health; cholesterol analog; cost; gramicidin A; insight; interest; magnetic field

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 2D ELDOR is a pulsed ESR technique that supplies greatly increased resolution to motional dynamics and local structure of biomolecules, such as membrane lipids and proteins, compared to conventional cw ESR techniques. In a recent study of the effect of the ion-channel forming peptide, gramicidin A, (GA) on model membrane structures, we could clearly distinguish and study the properties of both the boundary lipids that coat GA and the bulk lipids. That study at 17 GHz, however, indicated that higher frequency studies are needed, due to the increased orientational resolution they can provide. This enables better discrimination of the details of the ordering and dynamics. We have now extended the capabilities of our new high-power pulsed 95 GHz spectrometer to enable the study of spin-labeled lipids, such as the cholesterol analogue, CSL, and spin-labeled GA (GASL). The great challenges at 95 GHz are to have a sufficiently wide spectral coverage (as much as 350 MHz) to obtain the full nitroxide spin-label spectrum, and to have sufficiently short dead-times in view of the shortened T2 relaxation times at higher frequencies. We have succeeded, in particular, with macroscopically aligned membrane samples. Spectra have been obtained from GASL in aligned DPPC membranes just below room temperature. Using the new microtome technique, it was possible to obtain spectra for several orientations of the membrane normal with respect to the dc magnetic field. The parallel orientation shows the predominance of the molecular z- orientation, consistent with the way the GA aligns in DPPC. When the membrane is tilted perpendicular to the dc magnetic field, spectral contributions from the molecular x and y orientations become more pronounced as expected for such an aligned sample. What is of particular interest is that the 2D-ELDOR spectral patterns change with mixing time, Tm. This is a clear indication that these 2D-ELDOR spectra are showing slow-motional effects. That is, the various dynamic spin packet contributions to the spectrum are relaxing at different rates, leading to significant spectral changes with Tm. These results are expected to provide insight into the detailed dynamics of GA in the membranes.

该副本是利用资源的众多研究子项目之一 由NIH/NCRR资助的中心赠款提供。对该子弹的主要支持 而且，副投影的主要研究员可能是其他来源提供的 包括其他NIH来源。 列出的总费用可能 代表subproject使用的中心基础架构的估计量， NCRR赠款不直接向子弹或副本人员提供的直接资金。 2D Eldor是一种脉冲ESR技术，与常规的CW ESR技术相比，生物分子的运动动力学和局部结构（例如膜脂质和蛋白质）大大增加了分辨率。在最近对形成肽gramicidin A（GA）对模型膜结构的肽的影响的研究中，我们可以清楚地区分和研究涂层GA和大量脂质的边界脂质的性质。然而，该研究在17 GHz的研究表明，由于它们可以提供的方向分辨率增加，需要更高的频率研究。这可以更好地歧视订购和动态的细节。现在，我们扩展了新型高功率脉冲95 GHz光谱仪的能力，以便研究自旋标记的脂质，例如胆固醇类似物，CSL和自旋标记的GA（GASL）。 95 GHz面临的巨大挑战是要获得足够宽的频谱覆盖率（多达350 MHz），以获得完整的氮氧化型自旋标签光谱，并且鉴于在较高频率下T2放松时间缩短的缩短时间。我们特别是宏观对齐的膜样品成功了。从室温以下的对齐的DPPC膜中的GASL获得了光谱。使用新的微型机构技术，可以获得相对于直流磁场的膜法线的几个方向的光谱。平行方向显示了分子z-取向的优势，与GA在DPPC中对齐的方式一致。当将膜垂直于直流磁场倾斜时，分子X和Y方向的光谱贡献变得更加明显，如这种对齐样品所预期的。特别有趣的是，随着混合时间的混合时间，2D-ELDOR光谱模式变化。这清楚地表明，这些2D-ELDOR光谱正在显示慢电位效应。也就是说，各种动态自旋数据包对光谱的贡献以不同的速度放松，从而导致TM的频谱发生重大变化。这些结果有望洞悉膜中GA的详细动态。