PROTEIN ASSEMBLIES AND METALLOPROTEINS

蛋白质组装体和金属蛋白质

• 批准号: 7960414
• 负责人: Tatyana Polenova
• 金额: $ 20.34万
• 依托单位: UNIVERSITY OF DELAWARE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7960414
• 关键词: Biocompatible Materials; Chloride Peroxidase; Computer Retrieval of Information on Scientific Projects Database; Data; Funding; Grant; Hand; Institution; Investigation; Label; Metalloproteins; Molecular; NMR Spectroscopy; Proteins; Protocols documentation; Research; Research Personnel; Resolution; Resources; Role; Scheme; Solid; Solutions; Source; Structure; Substrate Specificity; United States National Institutes of Health; Vanadium; Vertebral column; X-Ray Crystallography; design; research study; solid state; solid state nuclear magnetic resonance; two-dimensional

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. PROJECT VI: Polenova - Project will investigate structure and conformational dynamics of a 68 kDa- vanadium chloroperoxidase in the solid state using multidimensional NMR spectroscopy. The focus on two major objectives: 1) determine the tertiary structure of chloroperoxidase using solid-state NMR data only; 2) to establish the multiple timescale conformational dynamics in the protein. To accomplish these objectives, three specific aims will be pursued: 1) 13C and 15N resonance assignments of uniformly labeled vanadium chloroperoxidase; 2) determination of long-range tertiary constraints and structure calculation; 3) investigation of multiple timescale conformational dynamics in uniformly and sparsely isotopically enriched protein. These studies would on one hand help elucidate the role of internal conformational dynamics in the substrate specificity of the vanadium haloperoxidases, and on the other, expand the capabilities of solids NMR to structural characterization of large noncrystalline proteins currently intractable by X-ray crystallography or solution NMR. Experimental plan. We completed the specific aim 1 and made significant progress for aim 2. The following experiments were conducted first: 1) heteronuclear 13C-15N two-dimensional correlation experiments to establish backbone resonance assignments, using the double cross polarization (DCP) mixing scheme;1 2) homonuclear two-dimensional 13C-13C correlation experiments to establish the sidechain assignments, using RFDR2 and RAD3 mixing schemes. In the possible case of the insufficient spectral resolution, the three-dimensional NCC protocols were pursued, involving NCACO and NCACB band-selective magnetization transfer schemes. With these experiments in hand, we have to assigned most of the resonances in the protein.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 项目六：Polenova -项目将使用多维NMR光谱研究固态68 kDa-钒氯过氧化物酶的结构和构象动力学。 重点是两个主要目标：1）确定的三级结构的氯过氧化物酶仅使用固态NMR数据; 2）建立多时间尺度的构象动力学的蛋白质。 为了实现这些目标，将追求三个具体的目标：1）13 C和15 N共振分配的均匀标记的钒氯过氧化物酶; 2）确定远程三级约束和结构计算; 3）调查的多时间尺度构象动力学均匀和稀疏同位素富集的蛋白质。 这些研究将一方面有助于阐明钒卤代过氧化物酶的底物特异性的内部构象动力学的作用，另一方面，扩大固体NMR的能力，目前难以通过X射线晶体学或溶液NMR的大型非结晶蛋白质的结构表征。 实验计划。 我们完成了具体目标1，并在目标2方面取得了重大进展。 首先进行以下实验：1）使用双交叉极化（DCP）混合方案，异源13 C-15 N二维相关实验以建立主链共振分配; 12）使用RFDR 2和RAD 3混合方案，同源二维13 C-13 C相关实验以建立侧链分配。 在光谱分辨率不足的可能情况下，追求三维NCC协议，涉及NCACO和NCACB带选择磁化转移方案。 有了这些实验，我们必须分配蛋白质中的大部分共振。