NEW SOLID STATE NMR METHODOLOGY FOR STRUCTURAL STUDIES OF BIOPOLYMERS

用于生物聚合物结构研究的新固态核磁共振方法

• 批准号: 6432095
• 负责人: ROBERT TYCKO
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6432095
• 关键词: HIV envelope protein gp120; conformation; glycerol; immunoconjugates; immunoglobulin structure; method development; nuclear magnetic resonance spectroscopy; peptide structure; structural biology; urea; water solution

In the past year, we have made substantial progress in the following two areas:1. Development of Multiple Quantum Carbon-13 Solid State NMR and Application to beta-Amyloid Fibril Structure We have developed solid state NMR techniques that allow the detection of multiple quantum NMR signals in carbon-13-labeled biopolymer samples. These multiple quantum signals arise from collective transitions of groups of dipole- coupled carbon-13 nuclear spins. Observation of multiple quantum signals requires that the carbon-13 nuclei be within approximately 5 angstroms of one another. Therefore, multiple quantum spectra can be used as a probe of biopolymer conformation or intermolecular packing in noncrystalline solids. We have applied the new multiple quantum NMR techniques in structural studies of fibrils formed by the 40-residue beta-amyloid peptide of Alzheimers diseases, as well as fibrils of shorter model peptides derived from the full-length beta-amyloid peptide. Comparison of experimental multiple quantum signal intensities with numerical simulations indicates unambiguously that the beta- amyloid peptides are arranged in a parallel beta-sheet structure in the fibrils. These results rule out existing structural models for beta- amyloid fibrils that are based on an anti-parallel arrangement. 2. Development of Multidimensional Solid State NMR Techniques for Structural Studies of Uniformly Labeled, Oriented Peptides and Proteins We have developed two- and three-dimensional NMR spectroscopic techniques that allow the resolution and sequential assignment of NMR signals in uniformly carbon-13 and nitrogen-15 labeled peptides and proteins that are uniaxially oriented with respect to the magnetic field of the solid state NMR spectrometer. These techniques overcome major hurdles to the determination of full structures of peptides and proteins, including membrane-bound peptides and proteins, by solid state NMR. The new techniques depend on novel homonuclear and heteronuclear decoupling sequences that permit the observation of carbon-13 and nitrogen-15 chemical shifts in the presence of strong dipole-dipole couplings that would otherwise render the spectra intractable. The new techniques have been demonstrated on simple model peptides in single crystal form. Extensions to bona fide proteins are in progress. - nuclear magnetic resonance, protein structure

一年来，我们在以下两方面取得了实质性进展：我们已经开发了固态核磁共振技术，可以检测碳-13标记生物聚合物样品中的多个量子核磁共振信号。这些多重量子信号来自偶极耦合碳-13核自旋群的集体跃迁。多重量子信号的观测要求碳-13原子核之间的距离在5埃以内。因此，多重量子光谱可以用作生物聚合物构象或非结晶固体分子间堆积的探针。我们将新的多重量子核磁共振技术应用于阿尔茨海默病40残基β -淀粉样肽形成的原纤维结构研究，以及由全长β -淀粉样肽衍生的较短模型肽的原纤维结构研究。实验多重量子信号强度与数值模拟的比较明确地表明β -淀粉样肽在原纤维中以平行的β -片结构排列。这些结果排除了现有的基于反平行排列的β -淀粉样蛋白原纤维结构模型。2. 我们已经开发了二维和三维核磁共振波谱技术，允许在碳-13和氮-15均匀标记的肽和蛋白质中进行核磁共振信号的分辨率和顺序分配，这些肽和蛋白质相对于固态核磁共振波谱仪的磁场单轴定向。这些技术克服了固态核磁共振测定肽和蛋白质（包括膜结合肽和蛋白质）完整结构的主要障碍。新技术依赖于新的同核和异核解耦序列，这些序列允许在强偶极子-偶极子耦合存在的情况下观察碳-13和氮-15的化学位移，否则将使光谱难以处理。新技术已经在单晶形式的简单多肽模型上得到了验证。对真正蛋白质的扩展正在进行中。-核磁共振，蛋白质结构