Methods/Measuring/Structural Restraints in Large Helical Proteins & Protein Comp

大螺旋蛋白的方法/测量/结构限制

• 批准号: 8260526
• 负责人: JAMES Jeiwen CHOU
• 金额: $ 23.97万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至 2013-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8260526
• 关键词: Amides; Amino Acids; Applied Research; Bacteriophage Pf1; Buffers; Collaborations; Complex; Coupling; DNA; Detection; Detergents; Development; Elements; Engineering; Entropy; Goals; High temperature of physical object; Hydrogen Bonding; Length; Measurement; Measures; Membrane Proteins; Methods; Molecular; NOESY; Nanostructures; Nanotechnology; Nanotubes; Phase; Physiologic pulse; Proteins; Protocols documentation; Relaxation; Research; Residual state; Resistance; Sampling; Scheme; Sodium Chloride; Structure; System; Testing; Time; Translation Initiation; Universities; Urea; Variant; Vertebral column; Water; Work; base; computer studies; design; experience; improved; liquid crystal; medical schools; optimal control theory; polyacrylamide gels; programs; protein complex; protein protein interaction; reconstruction; research study; restraint; structural biology; success; surfactant; tool

The overall goal of this component is to develop and test new alignment medium for dipolar coupling measurement and new strategies for obtaining structural restraints in large proteins and protein complexes. In the first phase of this component, we will focus on developing DMA nanostructures for weakly orienting protein complexes that are solubilized in harsh buffer conditions. Residual dipolar couplings (RDCs) are crucial experimental restraints in structure determination of large proteins when long-range NOEs are insufficient. RDCs are also very useful in orienting domains within protein complexes. Based on our initial success in making DNA nanotubes that can form stable liquid crystal in the presence of high concentration of detergents, we will establish a robust protocol for aligning detergent-protein complexes. The membrane protein systems to be tested include a number of ~30 kDa a helical, polytopic membrane proteins. We will also test the alignment of the EIF4e-4g complex, an important translation initiation interaction previously studied by Wagner and colleagues. This protein complex can only reach NMR-feasible concentration in the presence of a detergent known as CHAPS. In the second phase, we will develop methods of measuring structural restraints for large helical proteins. Hydrogen bond length has very small variation and serves as extremely stringent distance constraint for defining secondary structures. Measurement of the small 3JNCg couplings is perhaps the most straightforward and unambiguous method for determining protein sidechain xi angles. We will implement the relaxation optimized pulse elements (ROPE) to enable measurement of 3hJNc' across hydrogen bonds and 3JNCg that defines sidechain rotamer for large deuterated proteins. We will also develop 4D NOESY and 13C detection scheme for resolving resonance overlap. For helical proteins larger than 300 amino acids, it is usually not possible to resolve most of the short-range NOEs in the conventional15N-separated NOESY due to strong overlap of amide resonances. However, they can be resolved in a 4D 15N- and 13C'- edited NOESY (NOESY- HNCO). We will work with Jeff Hoch's group to significantly shorten the experimental time of 4D NOESY- HNCO by implementing nonuniform sampling and maximum entropy reconstruction methods so that it can be of practical use. We will also develop 13C-observation strategies for resolving resonance overlap and apply them to the measurements of 15N relaxation rates and RDCs.

该组件的总体目标是开发和测试用于偶极耦合的新对准介质 获得大蛋白质和蛋白质复合物结构限制的测量和新策略。 在该组件的第一阶段，我们将重点开发用于弱取向的 DMA 纳米结构 在苛刻的缓冲条件下溶解的蛋白质复合物。残余偶极耦合 (RDC) 是 当长程 NOE 存在时，大蛋白结构测定中的关键实验限制 不足的。 RDC 在蛋白质复合物内的结构域定向方面也非常有用。根据我们最初的 成功制造出在高浓度 DNA 存在下可形成稳定液晶的 DNA 纳米管 去污剂，我们将建立一个强大的方案来对齐去污剂-蛋白质复合物。膜 待测试的蛋白质系统包括许多约 30 kDa 的螺旋、多胞膜蛋白。我们将 还测试了 EIF4e-4g 复合物的比对，这是之前重要的翻译起始相互作用 瓦格纳及其同事进行了研究。该蛋白质复合物只能在 NMR 中达到可行的浓度 存在称为 CHAPS 的清洁剂。 在第二阶段，我们将开发测量大螺旋蛋白结构限制的方法。 氢键长度变化非常小，对氢键长度有极其严格的距离约束 定义二级结构。小型 3JNCg 联轴器的测量可能是最直接的 以及确定蛋白质侧链 xi 角度的明确方法。我们将实施放宽措施 优化的脉冲元件 (ROPE) 可测量跨氢键的 3hJNc' 和 3JNCg 定义了大氘代蛋白质的侧链旋转异构体。我们还将开发4D NOESY和13C检测 解决共振重叠的方案。对于大于 300 个氨基酸的螺旋蛋白，通常不是 由于强，可以解决传统15N分离NOESY中的大多数短程NOE 酰胺共振的重叠。然而，它们可以在 4D 15N- 和 13C'- 编辑的 NOESY (NOESY- HNCO）。我们将与Jeff Hoch团队合作，大幅缩短4D NOESY的实验时间- HNCO通过实施非均匀采样和最大熵重建方法，使其能够 具有实际用途。我们还将开发 13C 观测策略来解决共振重叠和 将它们应用于 15N 弛豫率和 RDC 的测量。