REFOLDING OF CARBONIC ANHYDRASE

描述：了解蛋白质折叠和再折叠是关键， 基因工程蛋白的最佳用途。 先前的研究表明 当碳酸酐酶变性并重新折叠时， 虽然看起来有一些小的变化，但已经恢复了。 ... 许多 重折叠蛋白质的测试检查整体结构和功能。 是 建议检查催化位点或附近的详细变化。 Co（II） 可以取代天然Zn（II）在碳的活性位点 脱水酶 建议使用Co（II）作为光谱探针， 重折叠前后的碳酸酐酶。 脉冲EPR技术将 用于分析Co（II）与三个组氨酸的相互作用， 金属结合位点，并检查金属位点的水合作用。 Co（II）与α-Al_2O_3之间电子-电子自旋相互作用的变化 硝酰基自旋标记作为抑制剂或 共价连接到人碳链中的单个半胱氨酸残基上， 脱水酶将用于探测更长范围的构象变化。 的 再折叠前酶的数据将与X射线衍射数据进行比较。 晶体学数据，以验证实验 方法. 本研究中采用的技术应适用于 其他含Zn（II）的蛋白质，已经表明Co（II）可以 在保留催化活性的情况下取代Zn（II）。 .

DESCRIPTION: An understanding of protein folding and re-folding is key to optium use of genetically engineered proteins. Previous studies have shown that when carbonic anhydrase is denatured and re-folded, catalytic activity is regained, although there appear to be some small changes. ... Many tests of refolded proteins examine overall structure and function. It is proposed to examine detailed changes at or near the catalytic site. Co(II) can be substituted for the native Zn(II) at the active site of carbonic anhydrase. It is proposed to use Co(II) as a spectroscopic probe of carbonic anhydrase before and after re-folding. Pulsed EPR techniques will be used to analyze the interaction of the Co(II) with three histidines at the metal-binding site and to examine the hydration of the metal site. Changes in electron-electron spin-spin interaction between Co(II) and a nitroxyl spin label added to the protein either as an inhibitor or covalently attached to the single cysteine residue in human carbonic anhydrase will be used to probe longer-range conformational changes. The data for the enzyme before re-folding will be compared with X-ray crystallographic data in the literature to validate the experimental methods. The techniques employed in this study should be applicable to other Zn(II)-containing proteins for which it has been shown that Co(II) can replace the Zn(II) with retention of catalytic activity ... .