FOLDING OF CARBONIC ANHYDRASE

描述：（改编自申请人的摘要）蛋白质折叠是一个主要的 当前感兴趣的话题。在这些研究中， 蛋白质内的局部环境和确定距离的技术 在蛋白质的区域之间。我们建议测试和校准两个EPR为基础的 技术使用牛和人的碳酸酐酶作为模型系统。1)的 结合在蛋白质活性部位的Co（II）离子的快速松弛效应 硝酰基自旋标记的电子自旋晶格弛豫速率 抑制剂或蛋白质的半胱氨酸残基将用于 确定金属结合位点与金属结合位点的位置之间的距离。 自旋标签。2)邻近甲基对自旋回波的影响 硝酰基自旋标记的退相速率将用于表征 Spin标签的环境。这两种技术将测试自旋标签 在正常折叠的碳酸酐酶中的已知位置。这些技术将 然后用于表征折叠中间和展开状态， 当盐酸胍加入到人碳酸酐酶中时， 二.预计这些技术将有助于其他研究。 自旋标记可以附着的蛋白质。

DESCRIPTION: (adapted from applicant's abstract) Protein folding is a major topic of current interest. In these studies it is useful to have probes of local environment within the protein and techniques to determine distances between regions of the protein. We propose to test and calibrate two EPR-based techniques using bovine and human carbonic anhydrase as model systems. 1) The effect of a rapidly relaxing Co(II) ion bound at the active site of the protein on the electron spin-lattice relaxation rate of a nitroxyl spin label attached to an inhibitor or to a cysteine residue of the protein will be used to determine the distance between the metal binding site and the location of the spin label. 2) The effect of neighboring methyl groups on the spin echo dephasing rate for a nitroxyl spin label will be used to characterize the environment of the spin label. Both techniques will be tested with spin labels at known locations in normally folded carbonic anhydrase. These techniques will then be used to characterize the folding intermediate and unfolded state that are observed when guanidine hydrochloride is added to human carbonic anhydrase II. It is anticipated that these techniques will be useful in studies of other proteins to which the spin labels can be attached.