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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 2D ELDOR是一种脉冲ESR技术，与传统的cw ESR技术相比，它大大提高了对生物分子（如膜脂质和蛋白质）的运动动力学和局部结构的分辨率。在最近的一项研究中的离子通道形成肽，短杆菌肽A，（GA）对模型膜结构的影响，我们可以清楚地区分和研究的性质的边界脂质涂层GA和散装脂质。然而，在17 GHz的研究表明，需要更高的频率研究，因为它们可以提供更高的取向分辨率。这使得能够更好地区分排序和动态的细节。我们现在已经扩展了我们新的高功率脉冲95 GHz光谱仪的功能，使自旋标记的脂质，如胆固醇类似物，CSL和自旋标记的GA（GASL）的研究。在95 GHz下的巨大挑战是具有足够宽的光谱覆盖（多达350 MHz）以获得完整的氮氧自由基自旋标记光谱，并且鉴于在较高频率下缩短的T2弛豫时间而具有足够短的死区时间。我们已经取得了成功，特别是宏观对齐的膜样品。光谱已获得从GASL在对齐的DPPC膜刚好低于室温。使用新的切片机技术，它是可能的，以获得光谱的几个方向的膜正常相对于直流磁场。平行取向显示了分子z-取向的优势，与DPPC中GA对齐的方式一致。当膜垂直于直流磁场倾斜时，来自分子x和y取向的光谱贡献变得更加明显，如对于这样的对准样品所预期的那样。特别感兴趣的是，2D-ELDOR光谱图案随着混合时间Tm而变化。这清楚地表明这些2D-ELDOR光谱显示出慢动效应。也就是说，对光谱的各种动态自旋包贡献以不同的速率弛豫，导致随Tm的显著光谱变化。这些结果预计将提供深入了解GA在膜中的详细动力学。