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.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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(如膜脂和蛋白质)的运动动力学和局部结构的分辨率。在最近关于离子通道形成肽甘草素A(GA)对模型膜结构的影响的研究中，我们可以清楚地区分和研究覆盖GA的边界脂和主体脂的性质。然而，17 GHz的研究表明，由于它们可以提供更高的定向分辨率，因此需要更高的频率研究。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，使自旋标记脂质的研究成为可能，例如胆固醇类似物、CSL和自旋标记GA(GASL)。在95 GHz的巨大挑战是具有足够宽的光谱覆盖(高达350 MHz)以获得完整的氮氧化物自旋标记谱，并且鉴于在较高频率下缩短的T2弛豫时间，具有足够短的死时间。特别是在宏观取向的膜样品方面，我们取得了成功。在室温以下的定向DPPC膜中从GASL中获得了光谱。使用新的显微切割机技术，可以获得膜法线相对于直流磁场的几个方向的光谱。平行取向显示了分子z取向的优势，这与GA在DPPC中的排列方式一致。当薄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，正如预期的那样，这种排列的样品。特别令人感兴趣的是，2D-Eldor光谱模式随混合时间TM而变化。这是一个明确的迹象，表明这些2D-Eldor光谱正在显示出慢运动效应。也就是说，各种动态自旋包对光谱的贡献以不同的速率驰豫，导致光谱随TM发生显著变化。这些结果有望提供对GA在膜中的详细动力学的洞察。