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.

我们已经扩展了在弱排列条件下研究溶液中大分子结构的技术。新的发展集中在蛋白质结构的组装上，该蛋白质结构从偶极耦合数据中，对NOES的1H-1H距离约束最低要求。已经开发了一个可靠的过程，该过程搜索了晶体学数据库的小片段，这些片段与实验观察到的偶性耦合匹配。这种所谓的分子片段替换（MFR）方法用于确定γ-S晶体的溶液结构，尽管链接两个域的肽序列的灵活性，但该结构的两个同源域彼此紧密地固定在彼此之间。 已经开发了一种程序，该程序允许将小角度X射线散射数据直接掺入NMR结构确定。虽然计算昂贵，但使用“地球仪？将某些肽组视为固定单点单位的方法，在完整的原子计算中，该方法通过三个数量级加速。在将实验性SAXS数据掺入结构细化后，应用于γ-S Crystallin在同源X射线结构上表现出相当大的拟合。预计SAXS数据的使用对于研究分子复合物的研究以及在溶液条件下研究复合系统的第四纪结构特别有用。 在洗涤剂胶束存在下α-核蛋白的研究表明，该蛋白采用α-螺旋构象，从残基10到92中采用α-螺旋构象，在残基40和44之间的转弯区域，以及残基动态无序的尾部区域99-140。当结合到降低弯曲的磷脂囊泡或突触表面时，与洗涤剂胶束结合时看到的U形构象可能存在，也可能不存在，尽管转弯区域的结构化特征表明可能是可能的结构作用。与帕金森氏病有关的两个突变体A30p和A56T的分析均与野生型相比，均显示A56T的结构变化，但脱离了A30p的第一个螺旋，从残基28开始，第二个螺旋中没有变化。第一个螺旋的变化降低了其曲率，从而影响了洗涤剂胶束的形状，这是第二螺旋在非常小的1H化学位移变化方面所感受到的。