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利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 项目六：Polenova-项目将使用多维核磁共振光谱研究固体中68 kDa钒氯过氧化物酶的结构和构象动力学。重点放在两个主要目标上：1)仅使用固体核磁共振数据确定氯过氧化物酶的三级结构；2)建立蛋白质中的多时间尺度构象动力学。为了实现这些目标，将追求三个特定的目标：1)统一标记的钒氯过氧化物酶的13C和15N共振指定；2)长程三级限制条件的确定和结构计算；3)研究均匀和稀疏同位素浓缩蛋白质的多时间尺度构象动力学。这些研究一方面将有助于阐明内部构象动力学在钒卤代过氧化物酶底物专一性中的作用，另一方面将扩大固体核磁共振的能力，以表征目前通过X射线结晶学或溶液核磁共振难以处理的大型非晶态蛋白质的结构。 实验计划。我们完成了特定的目标1，并对目标2取得了重大进展。首先进行了以下实验：1)异核~(13)C-15N二维关联实验，采用双交叉极化(DCP)混合方案；1)同核二维~(13)C-13C关联实验，建立侧链归属，使用RFDR2和Rad3混合方案。在光谱分辨率可能不足的情况下，采用三维NCC方案，包括NCACO和NCACB带选择性磁化转移方案。有了这些实验，我们必须指定蛋白质中的大多数共振。