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.

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