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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 项目 VI：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。 重点关注两个主要目标：1）仅使用固态 NMR 数据确定氯过氧化物酶的三级结构； 2）建立蛋白质的多时间尺度构象动力学。 为了实现这些目标，将追求三个具体目标：1）统一标记的钒氯过氧化物酶的 13C 和 15N 共振分配； 2）远程三级约束的确定和结构计算； 3）研究均匀和稀疏同位素富集蛋白质的多时间尺度构象动力学。 这些研究一方面有助于阐明内部构象动力学在钒卤过氧化物酶底物特异性中的作用，另一方面，将固体核磁共振的能力扩展到目前 X 射线晶体学或溶液难以处理的大型非晶体蛋白质的结构表征核磁共振。 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。首先进行了以下实验：1）异核 13C-15N 二维相关实验，使用双交叉极化（DCP）混合方案建立骨干共振分配；1 2)同核二维13C-13C相关实验建立侧链分配，使用RFDR2和RAD3混合方案。 在光谱分辨率可能不足的情况下，采用了三维NCC协议，包括NCACO和NCACB带选择磁化转移方案。 通过这些实验，我们必须分配蛋白质中的大部分共振。