REVEALING STRUCTURE DYNAMICS OF NEUROGLOBIN USING X-RAY SOLUTION SCATTERING A

使用 X 射线溶液散射 A 揭示神经球蛋白的结构动力学

• 批准号: 8363677
• 负责人: HYOTCHERL IHEE
• 金额: $ 4.86万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8363677
• 关键词: Amino Acid Sequence; Binding; Brain; Funding; Genetic Recombination; Globin; Grant; Heme; Heme Iron; Hemoglobin; Heterogeneity; Human; Molecular Conformation; Motion; Mus; Myoglobin; National Center for Research Resources; Optics; Principal Investigator; Process; Proteins; Reaction; Research; Research Infrastructure; Resolution; Resources; Roentgen Rays; Shapes; Slide; Solutions; Source; Spectrum Analysis; Structure; Techniques; Time; United States National Institutes of Health; Vertebrates; cost; flash photolysis; member; neuroglobin; research study; structural biology; time use

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Neuroglobin (Ngb) (Fig. 1) predominantly expressed in brain of humans and other vertebrate is a recently identified member of the globin superfamily. Despite a low amino acid sequence identity of about 25% and below the protein displays all the key determinants of canonical 3 over 3 alpha-helical globin fold. Phyllogenetic analysis revealed that Ngb forms a class distinct from the Myoglobin and Hemoglobin of vertebrates establishing its ancient origin. Crystal structure of murine ferric Ngb at 1.5 Angstrom resolution suggests significant heterogeneity of heme orientations (Fig. 2). The heme is observed to be inserted into the protein in two different orientations with an occupancy ratio of 70:30 (1 2). Spectroscopic studies of structural dynamics indicate that fluctuations between different conformations take place on longer time scales when compared to myoglobin. Flash photolysis experiments suggest that CO recombination process occurs on microsecond time scale (3). Moreover the structure of carbonmonoxy neuroglobin reveals substantial conformational changes upon binding of CO to the ferrous heme iron involving a sliding motion of the heme (45) also evident from the difference scattering curve calculated using Crysol (Fig. 3). Time-resolved optical spectroscopy has been used to study structural dynamics but the detailed structural fluctuations cannot be readily probed. In contrast time-resolved X-ray scattering techniques are sensitive to the structural fluctuations. For instance time-resolved small-angle X-ray scattering (TR-SAXS) is sensitive to global structural changes such as size and shape of a protein in solution while time-resolved wide-angle X-ray scattering (TR-WAXS) provides rich information about tertiary and quaternary structural changes of a protein with global sensitivity. Here we propose the time-resolved experiments on the two proteins murine neuroglobin and human myoglobin using TR-SAXS and TR-WAXS techniques to investigate the mechanism of photochemical reaction leading to the photocycle (Fig.4).

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 脑红蛋白（Neuroglobin，Ngb）是最近发现的一个球蛋白超家族成员，主要在人类和其他脊椎动物的脑中表达。 尽管具有约25%及以下的低氨基酸序列同一性，但该蛋白质显示出经典3个α-螺旋珠蛋白折叠超过3个α-螺旋珠蛋白折叠的所有关键决定因素。系统发育分析表明，Ngb形成了一个不同于脊椎动物的肌红蛋白和血红蛋白的类别，建立了其古老的起源。 在1.5埃分辨率下的鼠铁Ngb的晶体结构表明血红素取向的显著异质性（图2）。血红素被观察到以两种不同的方向插入蛋白质中，占有率为70：30（1 - 2）。结构动力学的光谱研究表明，不同构象之间的波动发生在较长的时间尺度相比，肌红蛋白。闪光光解实验表明，CO复合过程发生在微秒时间尺度上（3）。此外，一氧化碳神经红蛋白的结构揭示了当CO与亚铁血红素铁结合时的大量构象变化，包括血红素的滑动运动（45），这也从使用Crysol计算的差异散射曲线中显而易见（图3）。 时间分辨光谱已被用于研究结构动力学，但详细的结构波动不能很容易地探测。相反，时间分辨X射线散射技术对结构波动敏感。例如，时间分辨小角X射线散射（TR-SAXS）对溶液中蛋白质的大小和形状等全局结构变化敏感，而时间分辨广角X射线散射（TR-WAXS）提供了关于蛋白质的三级和四级结构变化的丰富信息，具有全局灵敏度。在这里，我们提出了时间分辨实验上的两种蛋白质，小鼠神经红蛋白和人肌红蛋白使用TR-SAXS和TR-WAXS技术，以研究光化学反应导致的光循环的机制（图4）。