Membrane Protein Solid State NMR: PISEMA Development and the M2 Tetramer Structure

膜蛋白固态 NMR：PISEMA 开发和 M2 四聚体结构

• 批准号: 0235774
• 负责人: Timothy Cross
• 金额: $ 51.63万
• 依托单位: Florida State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0235774&HistoricalAwards=false
• 关键词: Membrane; Protein; Solid; State; NMR

Membrane proteins represent approximately 30% of all genomes yet characterized. Today, less than 40 unique membrane protein structures are in the Protein Data Bank. Membrane proteins carry out numerous critical functions for cells and they are vastly different from their water-soluble counterparts through: their amino acid composition, the forces that stabilize their structure, their internal dynamics, etc. Sold state NMR is a demonstrated technology for achieving 3D high resolution structure in a lipid bilayer environment. This technology uniquely utilizes fully hydrated lipid bilayers for solvating protein samples. This work will further develop the primary solid state NMR experiment used for the structural characterization, PISEMA. Several modifications including the use of ramped pulses during the Lee-Goldburg sequence suggests significant gains in making PISEMA a more robust experiment. Advanced NMR probes with balanced RF circuits in high field magnets available at the NHMFL will be a great help in achieving this goal. The analysis of PISEMA spectra of alpha-helical proteins has led to cover stories in both the Journal of Magnetic Resonance and Protein Science. The characterization of solid state NMR data leads directly to a description of the tilt and rotational orientation of the helix within the lipid bilayer. Here, this analysis will be advanced using mathematical expressions to show how degeneracies in the structural solutions are resolved, by showing that unique spectral patterns are predicted for alpha, 310, and pi helices, and by characterizing the more complex spectral patterns for helices in the plane of the lipid bilayer. Moreover, it is the aim of this project to solve the complete backbone structure and assembly of the 44 kDa tetrameric M2 proton channel in hydrated lipid bilayers above the phase transition of the lipids. In this project Dr. Cross and his colleagues will develop a unique approach for determining the three dimensional structure of membrane proteins in their native environment - the lipid membrane that surrounds each cell. These proteins control everything that enters and leaves the cells, and they are responsible for accepting and sending information between cells. 30% or more of the proteins of a given genome are membrane proteins and yet technologies are very limited for their structural characterization. With a unique team of chemists, mathematicians and biophysicists, Dr. Cross brings together the necessary skills for the development of a Nuclear Magnetic Resonance methodology that has now been demonstrated to achieve unique structures of membrane proteins. Dr. Cross and his coworkers are refining the technology and enhancing its sensitivity for greater applications. This research will have a broad impact in both the fields of membrane protein biophysics and structural biology and students in chemical, biological and mathematical disciplines will be trained in a proven interdisciplinary environment.

膜蛋白约占所有已鉴定基因组的 30%。如今，蛋白质数据库中只有不到 40 个独特的膜蛋白结构。膜蛋白对细胞执行许多关键功能，它们与水溶性对应物有很大不同：它们的氨基酸组成、稳定其结构的力、它们的内部动力学等。固态 NMR 是一种实现 3D 的经过验证的技术脂质双层环境中的高分辨率结构。该技术独特地利用完全水合的脂质双层来溶解蛋白质样品。这项工作将进一步开发用于结构表征的初级固态核磁共振实验 PISEMA。一些修改，包括在 Lee-Goldburg 序列中使用斜坡脉冲，表明在使 PISEMA 成为一个更稳健的实验方面取得了显着的成果。 NHMFL 提供的高场磁体中配备平衡射频电路的先进 NMR 探头将为实现这一目标提供巨大帮助。对 α 螺旋蛋白 PISEMA 光谱的分析已成为《磁共振杂志》和《蛋白质科学》杂志的封面故事。固态核磁共振数据的表征直接导致对脂质双层内螺旋的倾斜和旋转方向的描述。在这里，将使用数学表达式推进该分析，通过显示预测 α、310 和 pi 螺旋的独特光谱模式，并通过表征脂质双层的平面。此外，该项目的目的是解决脂质相变上方水合脂质双层中 44 kDa 四聚体 M2 质子通道的完整主链结构和组装。 在这个项目中，克罗斯博士和他的同事将开发一种独特的方法来确定膜蛋白在其天然环境（包围每个细胞的脂质膜）中的三维结构。这些蛋白质控制着进入和离开细胞的一切，它们负责在细胞之间接受和发送信息。给定基因组中 30% 或更多的蛋白质是膜蛋白，但对其结构表征的技术非常有限。 Cross 博士拥有一支由化学家、数学家和生物物理学家组成的独特团队，汇集了开发核磁共振方法所需的技能，该方法现已被证明可以实现膜蛋白的独特结构。克罗斯博士和他的同事正在完善该技术并提高其灵敏度以实现更广泛的应用。这项研究将对膜蛋白生物物理学和结构生物学领域产生广泛影响，化学、生物和数学学科的学生将在经过验证的跨学科环境中接受培训。