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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 广角X射线溶液散射(WAXS)在表征蛋白质的结构、动力学和功能方面具有巨大的潜力，而不需要结晶(Fischetti等人，2003)。为了评估不同连接形式的血红蛋白在溶液中的结构之间的关系，收集了人正常成人一氧化碳-HbA(HbCO A)、在3 mM六磷酸肌醇(IHP)存在下的一氧化碳形式、二？-rHbCO(两个？链通过单个甘氨酸残基共价连接的变体)、重组rHbCO(？V96W-？N108K)以及MET和脱氧形式的牛Hb的溶液中的WAXS图谱。将观测到的模式与基于原子坐标集预测的模式进行了比较。我们的结论是，在溶液中，不同配位形式的Hb的结构与已知的晶体结构有明显的不同：(I)脱氧Hb与HbCO A在溶液中的结构差异比从晶体结构预测的要大。(2)MET-Hb在溶液中的结构比HbCO更接近脱氧Hb的结构。(3)Di-rHbCO的结构与HbCO A的结构非常相似。(4)IHP与HbCO A表面相距~43°的两个位置结合。根据它们的WAXS图谱对蛋白质浓度变化的响应：(I)脱氧-Hb的结构波动比HbCO A或Met-Hb大得多。(Ii)IHP导致HbCO A的结构起伏幅度略有增加。(Iii)二-rHbCO在溶液中没有表现出明显的结构起伏。(4)rHbCO(V96W/N108K)的波动幅度与HbCO A的波动幅度难以区分。

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.