Nuclear Magnetic Resonance--new Methods And Molecular St

核磁共振--新方法与分子研究

• 批准号: 7152050
• 负责人: Ad - Bax
• 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/7152050
• 关键词: X ray crystallography; alpha synuclein; calmodulin; chemical binding; conformation; intermolecular interaction; liquid crystal; magnetic field; method development; micelles; molecular polarity; nuclear magnetic resonance spectroscopy; nucleic acid structure; protein structure; structural biology

We have extended our technology for studying macromolecular structure in solution under weakly aligning conditions. New developments focus on the assembly of protein structure from dipolar coupling data with a minimum requirement for 1H-1H distance restraints from NOEs. A robust procedure has been developed that searches the crystallographic database for small fragments that match experimentally observed dipolar couplings. This so-called molecular fragment replacement (MFR) method was used to determine the solution structure of gamma-S crystalline, which was shown to have its two homologous domains tightly anchored to one another, despite flexibility in the peptide sequence linking the two domains. A procedure has been developed that permits direct incorporation of small angle X-ray scattering data into NMR structure determination. Although computationally expensive, use of a ?glob? approach, which treats certain peptide groups as fixed single point units, accelerates the method by three orders of magnitude over a full atom calculation. Application to gamma-S crystallin showed a considerably better fit to homologous X-ray structure upon incorporation of experimental SAXS data in the structure refinement. Use of SAXS data is anticipated to be particularly useful for the study of molecular complexes and for studying quaternary structure of complex systems under solution conditions. Study of alpha-synuclein in the presence of detergent micelles shows that this protein adopts an alpha-helical conformation from residue 10 through 92, with a turn region between residues 40 and 44, and a dynamically disordered tail region for residues 99-140. The U-shaped conformation seen when bound to a detergent micelle may or may not be present when bound to lower curved phospholipids vesicles or synapse surfaces, although the structured features of the turn region imply a possible structural role. Analysis of two mutants, A30P and A56T, both implicated in Parkinson?s disease show essentially no structural change for A56T compared to wild type, but an unraveling of the first helix of A30P, starting from residue 28, and no change in the second helix. The change in the first helix decreases its curvature and thereby impacts the detergent micelle shape, which is sensed by the second helix in terms of very small 1H chemical shift changes.

我们已经扩展了我们的技术，用于研究弱取向条件下溶液中的大分子结构。新的发展集中在组装蛋白质结构的偶极耦合数据与最低要求的1H-1H距离限制NOE。已经开发了一个强大的程序，搜索晶体数据库中的小片段，匹配实验观察到的偶极耦合。这种所谓的分子片段置换（MFR）方法用于确定γ-S晶体的溶液结构，该晶体显示其两个同源结构域彼此紧密锚定，尽管连接两个结构域的肽序列具有灵活性。 一个程序已经开发，允许直接纳入小角X射线散射数据到NMR结构测定。虽然计算昂贵，使用一个？一团？将某些肽基团视为固定单点单元的方法，在全原子计算上将该方法加速了三个数量级。应用γ-S晶体蛋白显示出相当好的适合同源X射线结构后，将实验SAXS数据的结构细化。预计使用SAXS数据是特别有用的分子配合物的研究和研究溶液条件下的复杂系统的四级结构。 在洗涤剂胶束存在下对α-突触核蛋白的研究表明，该蛋白质从残基10至92采用α-螺旋构象，在残基40和44之间具有转弯区域，并且残基99-140具有动态无序的尾区。当结合到洗涤剂胶束时看到的U形构象可能存在或可能不存在，当结合到较低的弯曲磷脂囊泡或突触表面时，尽管转弯区域的结构特征暗示了可能的结构作用。分析两个突变体，A30 P和A56 T，都牵连在帕金森？与野生型相比，S.S.疾病的A56 T基本上没有结构变化，但A30 P的第一螺旋从残基28开始解开，第二螺旋没有变化。第一螺旋的变化降低了其曲率，从而影响了洗涤剂胶束的形状，这是由第二螺旋在非常小的1H化学位移变化方面感测到的。