Oligomeric Structure of Membrane Peptides From Solid-State NMR

膜蛋白折叠涉及独立稳定的螺旋寡聚成适当的三级结构。关于蛋白质如何在膜中寡聚化的知识目前受到脂质双层中小寡聚肽的高分辨率结构的缺乏，特别是缺乏分子间结构限制的限制。该项目的第一个主要目标是开发固态NMR（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。魔角旋转下的1H驱动各向异性自旋扩散方法将用于确定肽的寡聚体数量。将分析自旋扩散的时间常数，以产生聚集体中不同分子之间的半定量距离。更多的定量距离将从异质偶极耦合中提取，特别是质子与低频异质自旋（如13 C和2 H）之间的耦合。质子的高旋磁比将扩展SSNMR的作用距离。 1H-2 H偶极再偶联将专门探讨，使分子间的界面，通过甲基氘代氨基酸侧链的研究。这种1H-X距离技术将被扩展，以允许更快的旋转速度，并提高1H homemandelsdecoupling的效率，从而能够测量更长的距离。随着这些方法的发展，两个同源寡聚螺旋束，甲型流感病毒M2蛋白（M2-TMP）的跨膜肽和设计的卷曲螺旋肽GCN 4-MS 1的结构将被研究。虽然这些肽的低聚状态在洗涤剂胶束中是已知的，但它们在脂质双层中还没有被直接测定。此外，没有分子间距离和包装信息是已知的。现在将使用19 F和13 C自旋扩散在脂质双层中直接测量聚集态。分子间的距离将提取均质自旋扩散曲线和异源偶极再耦合实验。此外，将研究聚集状态和螺旋束直径如何受到膜组成、膜厚度和氨基酸序列的影响。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来检查。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体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测定扩展到四级结构测定。它将加强对本科生、研究生和代表性不足群体的培训和教育。该研究的跨学科性质将使研究生受益，并促进化学，生物化学和生物物理学本科研究人员的招聘。将继续鼓励传统上积极参与PI研究的女学生和本科研究人员加入这项工作。通过这个项目获得的膜蛋白质折叠的知识将被纳入本科热力学的教学，如吉布斯自由能和化学平衡的主题。

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.