Oligomeric Structure of Membrane Peptides From Solid-State NMR

固态 NMR 的膜肽寡聚结构

• 批准号: 0543473
• 负责人: Mei Hong
• 金额: $ 68.74万
• 依托单位: Iowa State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-05-01 至 2011-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0543473&HistoricalAwards=false
• 关键词: Oligomeric; Structure; Membrane; Peptides; Solid

Membrane protein folding involves oligomerization of independently stable helices into proper tertiary structures. Knowledge on how proteins oligomerize in the membrane is currently limited by the paucity of high-resolution structures of small oligomeric peptides in lipid bilayers, in particular the lack of intermolecular structure restraints. The first major objective of this project is to develop solid-state NMR (SSNMR) techniques that directly determine the oligomeric number of peptides in lipid bilayers and that yield site-specific intermolecular distance constraints. A 1H-driven anisotropic spin diffusion method under magic-angle spinning will be used to determine the oligomeric number of peptides. The time constants of spin diffusion will be analyzed to yield semi-quantitative distances between different molecules in the aggregate. More quantitative distances will be extracted from heteronuclear dipolar couplings, especially the couplings between protons and low-frequency heteronuclear spins such as 13C and 2H. The high gyromagnetic ratio of the proton will extend the distance reach of SSNMR. 1H-2H dipolar recoupling will be specifically explored to enable the study of intermolecular interfaces through methyl-deuterated amino acid sidechains. This 1H-X distance technique will be extended to allow faster spinning speeds and to increase the efficiency of 1H homonuclear decoupling to enable the measurement of longer distances. With the development of these methods, the structure of two homo-oligomeric helical bundles, the transmembrane peptide of the M2 protein (M2-TMP) of the influenza A virus, and the designed coiled-coil peptide GCN4-MS1, will be investigated. Although the oligomeric states of these peptides are known in detergent micelles, they have not been directly determined in lipid bilayers. Moreover, no intermolecular distances and packing information are known. The aggregation states will now be directly measured in lipid bilayers using 19F and 13C spin diffusion. Intermolecular distances will be extracted both from homonuclear spin diffusion curves and from heteronuclear dipolar recoupling experiments. Furthermore, how the aggregation state and the helical bundle diameter are affected by the membrane composition, membrane thickness, and the amino acid sequence, will be studied. The effect of the size and polarity of the sidechain on the stability of the helical bundle will also be examined through sequence mutations. This project will have wide-ranging impacts on membrane biophysics, providing heretofore unavailable high-resolution structural information on membrane peptide assemblies. It will extend the frontier of NMR structural biology from secondary and tertiary structure determination to quaternary structure determination. It will enhance the training and education of undergraduate students, graduate students, and under-represented groups. The interdisciplinary nature of the research will benefit graduate students and facilitate the recruiting of undergraduate researchers in chemistry, biochemistry, and biophysics. Women students and undergraduate researchers, who have traditionally participated well in the PI's research, will continued to be encouraged to join the effort. Knowledge gained on membrane protein folding through this project will be incorporated into the teaching of undergraduate thermodynamics, on topics such as Gibbs free energy and chemical equilibrium.

膜蛋白折叠涉及独立稳定的螺旋寡聚化成适当的三级结构。目前，由于缺乏脂质双层中小寡聚肽的高分辨率结构，特别是缺乏分子间结构约束，关于膜中蛋白质寡聚的知识受到限制。该项目的第一个主要目标是开发固态核磁共振（SSNMR）技术，直接确定脂质双层中肽的寡聚体数量，并产生特定位点的分子间距离限制。采用魔角自旋下1h驱动各向异性自旋扩散法测定多肽的寡聚体数目。通过分析自旋扩散的时间常数，可以得到聚合体中不同分子之间的半定量距离。更多的定量距离将从异核偶极耦合中提取出来，特别是质子与低频异核自旋（如13C和2H）之间的耦合。质子的高回旋磁比将扩大SSNMR的距离范围。将专门探讨1H-2H偶极重偶联，以便通过甲基-氘化氨基酸侧链研究分子间界面。这种1H- x距离技术将得到扩展，以允许更快的旋转速度，并提高1H同核解耦的效率，从而能够测量更长的距离。随着这些方法的发展，将对甲型流感病毒M2蛋白的跨膜肽（M2- tmp）和设计的螺旋状肽GCN4-MS1的两个同源寡聚螺旋束的结构进行研究。虽然这些肽的寡聚状态在洗涤剂胶束中是已知的，但它们还没有在脂质双分子层中直接确定。此外，不知道分子间距离和包装信息。现在将使用19F和13C自旋扩散直接测量脂质双层中的聚集状态。分子间距离将从同核自旋扩散曲线和异核偶极重偶联实验中提取。此外，还将研究膜的组成、膜的厚度和氨基酸序列对聚集状态和螺旋束直径的影响。侧链的大小和极性对螺旋束稳定性的影响也将通过序列突变进行检验。该项目将对膜生物物理学产生广泛的影响，提供迄今为止无法获得的膜肽组装的高分辨率结构信息。它将核磁共振结构生物学的前沿从二级和三级结构的确定扩展到四级结构的确定。它将加强对本科生、研究生和弱势群体的培训和教育。这项研究的跨学科性质将有利于研究生，并促进招收化学、生物化学和生物物理学的本科生研究人员。传统上积极参与PI研究的女学生和本科研究人员将继续被鼓励参加这项工作。通过这个项目获得的关于膜蛋白折叠的知识将被纳入本科热力学的教学，包括吉布斯自由能和化学平衡等主题。