WAXS AS A PROBE FOR THE STUDY OF PROTEIN STRUCTURE, DYNAMICS AND FUNCTION

蜡作为研究蛋白质结构、动力学和功能的探针

• 批准号: 8361272
• 负责人: LEE MAKOWSKI
• 金额: $ 4.15万
• 依托单位: ILLINOIS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8361272
• 关键词: Adult; Binding; Biophysics; Cattle; Crystallization; Exhibits; Funding; Glycine; Grant; Hemoglobin; Human; Ligands; Link; National Center for Research Resources; Pattern; Phytic Acid; Principal Investigator; Proteins; Recombinants; Research; Research Infrastructure; Resources; Site; Solutions; Source; Structure; Surface; United States National Institutes of Health; Variant; base; cost; meetings; protein function; protein structure

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. Wide-angle x-ray solution scattering (WAXS) has significant potential for characterizing the structure, dynamics, and function of proteins without requiring crystallization (Fischetti et al., 2003). To assess the relationships among the structures of hemoglobins in different liganded forms in solution, WAXS patterns were collected from solutions of human normal adult carbonmonoxy-Hb A (HbCO A), the carbonmonoxy-form in the presence of 3 mM inositol hexaphosphate (IHP), di-¿-rHbCO (a variant in which the two ¿-chains are covalently linked by a single glycine residue), a recombinant rHbCO (¿V96W-¿N108K), and bovine Hb in the met- and deoxy- forms. Comparisons were made between the observed patterns and those predicted on the basis of atomic coordinate sets. We conclude that, in solution, the structures of Hb in different liganded forms exhibit clear differences from known crystal structures: (i) The structural difference between deoxy-Hb and HbCO A in solution is larger than predicted from the crystal structures. (ii) The structure of met-Hb in solution corresponds more closely to that of deoxy-Hb than HbCO. (iii) The structure of di-¿-rHbCO is very similar to that of HbCO A. (iv) IHP binds to two sites located ~43 ¿ apart on the surface of HbCO A. Based on the responsiveness of their WAXS patterns to changes in protein concentration: (i) Deoxy-Hb exhibits much larger structural fluctuations than HbCO A or met-Hb. (ii) IHP induces a modest increase in the magnitude of structural fluctuations in HbCO A. (iii) di-¿-rHbCO exhibits no observable structural fluctuations in solution. (iv) rHbCO (¿V96W/¿N108K) exhibits fluctuations of an amplitude indistinguishable from those of HbCO A.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供 该子项目的主要支持。 并且子项目的主要研究者可能是由其他来源提供的， 包括其他 NIH 来源的子项目可能列出的总成本。 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 广角 X 射线溶液散射 (WAXS) 具有无需结晶即可表征蛋白质结构、动力学和功能的巨大潜力（Fischetti 等人，2003）评估不同配体形式的血红蛋白结构之间的关系。在溶液中，WAXS 模式是从人类正常成人碳单氧血红蛋白 A (HbCO A) 的溶液中收集的，这是在 3 mM 肌醇六磷酸存在下的碳单氧形式（IHP），di-¿ -rHbCO（两条 β 链通过单个甘氨酸残基共价连接的变体）、重组 rHbCO (¡V96W-¡N108K) 以及甲硫氨酸和脱氧形式的牛 Hb 之间进行了比较。我们得出的结论是，在溶液中，不同配体形式的 Hb 结构与已知晶体表现出明显的差异。结构： (i) 溶液中的脱氧-Hb 和 HbCO A 之间的结构差异比晶体结构预测的要大 (ii) 溶液中的 met-Hb 的结构比 HbCO 更接近脱氧-Hb 的结构。 ) di-¿ 的结构-rHbCO 与 HbCO A 非常相似。 (iv) IHP 与位于 ~43 ¿ 的两个位点结合基于其 WAXS 模式对蛋白质浓度变化的响应：(i) Deoxy-Hb 表现出比 HbCO A 或 met-Hb 大得多的结构波动 (ii) IHP 导致适度增加。 HbCO A. (iii) di-¿ 结构性波动的幅度-rHbCO 在溶液中没有表现出可观察到的结构波动 (iv) rHbCO (¿V96W/¿N108K) 表现出的波动幅度与 HbCO A 的波动幅度无法区分。