CLEAVAGE MAPPING OF PROTEIN STRUCTURE

虽然许多小蛋白在体外可以自发折叠，但 折叠途径的鉴定、检测和结构 折叠中间体的表征一直很困难。最近， 人们的注意力已经转向熔化的球状状态和其他非本地人 被认为是动力学模型的蛋白质的平衡状态 蛋白质折叠的中间体。熔融的球状状态 去肌红蛋白在还原Ph时可以在平衡状态下聚集为 通过一些光学和流体力学方法进行了演示，并已 最近被证明是肌红蛋白上的一种动力学中间体 折叠路径。酰胺保护研究发现，只有 肌红蛋白的A、G和H螺旋在熔化的球体中受到保护 国家，表明涉及这些地区的类似原住民的下层结构 可能会持续下去。已经产生了葡萄球菌核酸酶的片段 它也具有与熔化的球体相似的性质，即 有点紧凑的结构，有一些二级结构，但没有 一个明确的三级结构。 最近我们开发了一种化学切割方法，其中EDTA-Fe 碱性试剂(EPD)可以通过半胱氨酸侧连接到蛋白质上 链条。抗坏血酸的加入会在铁上产生羟基自由基 在一个区域内扩散和切割多肽骨架的中心 靠近半胱氨酸结合部位的残基，溶剂可以接触到。 观察到的裂解位点可以通过氨基酸测序来定位。这个 切割依赖于蛋白质的构象。我们建议 无肌红蛋白和葡萄球菌熔融球状状态的表征 使用这种新开发的化学裂解的核酸酶片段结构 技术。我们将准备一些半胱氨酸的变种 蛋白质和表征在天然中观察到的切割模式 和熔化的球状状态。

While many small proteins can refold spontaneously in vitro, the identification of the folding pathway, and the detection and structural characterization of folding intermediates have been difficult. Recently, attention has turned to the molten globule state and other nonnative equilibrium states of proteins which are thought to be models for kinetic intermediates in protein folding. The molten globule state of apomyoglobin can be populated at equilibrium at reduced Ph as demonstrated by a number of optical and hydrodynamic methods, and has recently been shown to represent a kinetic intermediate on the myoglobin folding pathway. Amide protection studies have identified that only the A, G, and H helices of myoglobin are protected in the molten globule state, suggesting that a native-like substructure involving those regions may persist. Fragments of staphylococcal nuclease have been produced which also have properties similar to those of the molten globule, i.e. a somewhat compact structure with some secondary structure but without a defined tertiary structure. Recently we have developed a chemical cleavage method where an EDTA-Fe based reagent (EPD) can be attached to a protein via a cysteine side chain. The addition of ascorbate generates hydroxyl radicals at the iron center which diffuse and cleave the polypeptide backbone in a region close to the cysteine attachment site at residues accessible to solvent. The observed cleavage sites can be mapped by amino acid sequencing. The cleavage is dependent on protein conformation. We propose to characterize the molten globule state of apomyoglobin and staphylococcal nuclease fragment structures using this newly developed chemical cleave technique. We will prepare a number of cysteine variants of these proteins and characterize the cleavage patterns observed in the native and molten globule states.