CLEAVAGE MAPPING OF PROTEIN STRUCTURE

虽然许多小的蛋白质可以在体外自发地重折叠， 折叠途径的鉴定，以及 折叠中间体的表征一直是困难的。 最近， 人们的注意力已经转向了熔融球态和其他非原生态。 蛋白质的平衡状态被认为是动力学模型， 蛋白质折叠的中间体。 熔融球态的 脱辅基肌红蛋白可以在降低的pH下平衡地聚集， 通过一些光学和流体动力学方法证明， 最近被证明是肌红蛋白的动力学中间体 折叠途径 酰胺保护研究已经确定， 肌红蛋白的A、G和H螺旋在熔融球中受到保护 国家，这表明，一个类似本土的子结构，涉及这些地区 可能会持续下去。 葡萄球菌核酸酶的片段已经产生 其也具有与熔融球相似的性质，即， 有点紧凑的结构，有一些二级结构，但没有 明确的三级结构。 最近，我们开发了一种化学裂解方法，其中EDTA-Fe 基于EPD的试剂（EPD）可以通过半胱氨酸侧连接到蛋白质 链 抗坏血酸的加入在铁离子上产生羟基自由基， 在一个区域中扩散和切割多肽骨架的中心 在溶剂可接近的残基处靠近半胱氨酸附着位点。 观察到的切割位点可通过氨基酸测序作图。 的 切割取决于蛋白质构象。 我们建议 表征脱辅基肌红蛋白和葡萄球菌的熔融球状态 使用这种新开发的化学切割的核酸酶片段结构 法 我们将制备一些半胱氨酸变体， 蛋白质，并表征在天然细胞中观察到的切割模式。 和熔融球态。

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.