STUDIES OF CONFORMATIONAL CHANGES IN SOLUBLE GUANYLATE CYCLASE

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 可溶性鸟苷酸环化酶 (sGC) 是一种 150 kD 异二聚体蛋白，是细胞中主要的一氧化氮 (NO) 受体。 NO 与 β 亚基 N 端结构域中的血红素结合，刺激 cGMP 的产生并导致 NO 依赖性信号级联反应。 体外研究表明，配体结合时可能会发生变构构象变化。 除了生理配体 NO 之外，还有效应配体，以 YC-1 为代表，与一氧化碳 (CO) 协同作用。 我们要求光束时间对可溶性鸟苷酸环化酶进行小角度 X 射线散射研究，以表征配体结合相关的构象变化。 人类和天蛾 sGC 的几个不同结构域和构建体已通过重组方法表达并且可用，或将在不久的将来可用。 初步分析超速离心研究表明，构象变化足够大，足以通过 SAXS 进行表征，并且蛋白质在离心实验条件下不会显着聚集。

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. Soluble guanylate cyclase (sGC), a 150 kD heterodimeric protein, is the primary nitric oxide (NO) receptor in the cell. NO binds to heme in the N-terminal domain of the beta subunit, stimulates the production of cGMP and leads to NO-dependent signaling cascades. In vitro studies have shown that a probably allosteric conformational change takes place when ligands bind. In addition to the physiological ligand NO, there are effector ligands, typified by YC-1 that act in concert with carbon monoxide (CO). We request beam time to perform small angle x-ray scattering studies on soluble guanylate cyclase in order to characterize ligand binding associated conformational changes. Several different domains and constructs of both human and Manduca sexta sGC have been expressed by recombinant methods and are available, or will become available in the near future. Preliminary analytical ultracentrifugation studies demonstrate that the conformational change is large enough to be characterized by SAXS and that the protein does not aggregate significantly under the conditions of the centrifugation experiment.