IMPROVED TIME SHARED (TS) 3D-NOESY 13C,15N-HSQC FOR LARGE PROTEINS

针对大蛋白质改进的分时 (TS) 3D-NOESY 13C,15N-HSQC

• 批准号: 8361202
• 负责人: JOHN LUTE MARKLEY
• 金额: $ 0.37万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2012-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361202
• 关键词: Amides; Biological Preservation; Carbon; Data; Elements; Funding; Grant; Knowledge; Label; NOESY; National Center for Research Resources; Parents; Physiologic pulse; Principal Investigator; Proteins; Reporting; Research; Research Infrastructure; Resolution; Resources; Sampling; Scheme; Signal Transduction; Source; Time; United States National Institutes of Health; Water; base; carbene; cost; design; improved; methyl group; research study

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. It is common knowledge that approaches that saturate water magnetization will yield lower sensitivity for those resonances that exchange with water, namely amide groups in proteins. Thus, to achieve maximum sensitivity, experiments that involve amide groups, such as NOESY 15N-HSQC, have been designed to preserve water magnetization. We report here a modified approach for the simultaneous NOESY 13C,15N?HSQC experiment, based on the NOESY 15N-fast HSQC pulse sequence, that features a WATERGATE element for water suppression and preservation. The result is a simultaneous NOESY experiment that provides better water suppression, better spectral quality and sensitivity comparable to that achieved by the parent the 15N-NOESY spectrum acquired independently, but improved compared to a 13C- and to previously reported 13C,15N simultaneous approaches. The only drawback of our approach is that the water suppression scheme wipes out resonances near the water, most notably those from 13C? groups. Because of this, we optimize the 13C spectral window to achieve maximal signal and resolution from methylene and especially methyl groups. Given these features, our simultaneous experiment is ideally suited for collecting NOESY spectra in water with samples of medium to large size proteins that are U-2H, U-15N and methyl 13C,1H labeled.For U-15N and U-13C labeled protein samples, a second 3D experiment must be collected to get NOESY peaks from 13C? groups, and for this, we propose using a 13C-NOESY experiment based on the sensitivity-enhanced 13C-HSQC pulse sequence, with the carbon window optimized for 13C? groups. This approach yields good water suppression and good S/N for the 13C? groups, and the data can be acquired in less time than for a regular full aliphatic carbon NOESY experiment.

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