Development and Application of Solid State NMR for Biomo

2005财年在以下领域取得了进展：(1)我们继续进行淀粉样蛋白原纤维的结构研究。我们使用新的固态核磁共振方法，确定了阿尔茨海默病β -淀粉样肽形成的淀粉样原纤维中的一些四级接触。这使我们能够开发一种修正的β -淀粉样蛋白原纤维结构模型，其中结构的所有基本方面都是由实验数据确定的。这个模型适用于在温和搅拌下生长的原纤维。我们还开发了在静态条件下制备β -淀粉样蛋白原纤维的改进技术，使原纤维具有均匀的形态和干净的核磁共振光谱。这种可选择的纤维形式的结构表征正在进行中。我们也开始了由胰岛淀粉样前体蛋白（又名胰淀素）形成的原纤维的结构研究。在我们的胰淀素项目中，我们已经开发了合成和纯化肽的有效方案，以及制备具有统一形态和高质量固态核磁共振光谱的原纤维的方案。(2)我们继续对来自HIV-1 Vpu的40个残基肽进行结构研究，其中包含磷脂双层中的跨膜段。利用一种新型的二维固体核磁共振，我们已经确定了Vpu低聚物中跨膜螺旋段之间的分子间接触。这些接触使我们能够开发基于实验的低聚Vpu离子通道超分子结构模型。(3)我们开发了新的固态核磁共振方法，包括在含有均匀标记的氨基酸的样品中选择性测量碳-13标签之间特定原子间距离的新方法。这种选择性距离测量的新方法似乎可能促进固态核磁共振广泛的结构研究。此外，我们在具有魔角旋转能力的新型超低温固态核磁共振探针的设计、建造和初步演示方面取得了重大进展。这种新的固态核磁共振探头有望提高固态核磁共振测量的灵敏度10倍或更多。

Progress in FY 2005 has been in the following areas: (1) We have continued structural studies of amyloid fibrils. We have determined a number of quaternary contacts in amyloid fibrils formed by the Alzheimer's beta-amyloid peptide, using new solid state NMR methods. This allows us to develop a revised structural model for beta-amyloid fibrils, in which all basic aspects of the structure are determined by experimental data. This model applies to fibrils grown with gentle agitation. We have also developed improved techniques for preparing beta-amyloid fibrils under quiescent conditions, such that the fibrils have homogeneous morphologies and clean NMR spectra. Structural characterization of this alternative fibril form is under way. We have also begun structural studies of fibrils formed by the islet amyloid precursor protein (a.k.a. amylin). In our amylin project, we have developed efficient protocols for synthesizing and purifying the peptide, as well as protocols for preparing fibrils with uniform morphologies and high-quality solid state NMR spectra. (2) We have continued structural studies of a 40-residue peptide from HIV-1 Vpu, containing its transmembrane segment, in phospholipid bilayers. Using a novel form of two-dimensional solid state NMR, we have identified intermolecular contacts between transmembrane helical segments in Vpu oligomers. These contacts allow us to develop experimentally-based models for the supramolecular structure of oligomeric Vpu ion channels. (3) We have developed new solid state NMR methods, including a new approach to selective measurement of specific interatomic distances between carbon-13 labels in samples that contain uniformly-labeled amino acids. This new approach to selective distance measurements seems likely to facilitate a wide range of structural studies by solid state NMR. In addition, we have made significant progress on the design, construction, and initial demonstration of a new ultra-low-temperature solid state NMR probe with magic-angle spinning capabilities. This new solid state NMR probe promises to improve the sensitivity of solid state NMR measurements by a factor of 10 or more.