STRUCTURAL ANALYSIS OF PROTEIN FOLDING INTERMEDIATES

蛋白质折叠中间体的结构分析

• 批准号: 2022887
• 负责人: Robert O. Fox
• 金额: $ 21.24万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2000-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2022887
• 关键词: X ray crystallography; chemical cleavage; chemical kinetics; conformation; myoglobin; nuclear magnetic resonance spectroscopy; nuclease; protein folding; protein sequence; protein structure; structural biology

While may 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. 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. Pulsed hydrogen-deuterium exchange during refolding has been used to probe the protection of backbone amide hydrogens from solvent exchange during refolding of a number of proteins and most recently the staphylococcal nuclease Pro 117 yields Gly variant. The extent of exchange for 39 residues is determined by two-dimensional proton NMR after refolding for 5 ms to 10s. Three kinetic phases are inferred. Modest protection of amides in the early refolding intermediate composed to two beta-sheets formed by local sequence interactions is observed after a 5 ms refolding period. Native levels of protection throughout the molecule accrue more slowly in tow kinetic phases (k approximately 2s-1, k less than 0.01s-1). Protection factors were determined by varying the high pH labeling pulse after refolding for 100 ms. Little or no native or unfolded protein is present; instead, most molecules are in one or more partially folded states. The intermediate state has modest, yet significant, protection for residues in the beta-sheets (protection factors 10-60), and almost no protection in the alpha- helices (protection factors less than 10). The pattern of labeling is consistent with a role for beta turns and beta-hairpins in the formation of the early intermediate. Recently a chemical cleavage method has been developed where an EDT-Fe based reagent (EPD-Fe) 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 characterize the molten globule state of apomyoglobin and staphylococcal nuclease fragment structures using this newly developed chemical cleavage 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.

虽然可能的小蛋白质可以自发地体外重新分配 折叠途径的识别以及检测和结构 折叠中间体的表征很困难。 最近，注意力转向了熔融的球状态和其他 被认为是模型的蛋白质的非本地平衡状态 用于蛋白质折叠中的动力学中间体。 碎片 已经产生了葡萄球菌核酸酶，也具有特性 与熔融球的相似，即有点紧凑 结构具有一定的二级结构，但没有定义的第三结构 结构。 重折叠过程中的脉冲氢 - 脱欧交换已用于 探测骨干酰胺氢的保护免受溶剂交换 在重新折叠多种蛋白质期间，最近 葡萄球菌核酸酶Pro 117产生Gly变体。 程度 交换39个残基由二维质子NMR确定 重折5毫秒至10s之后。 推断三个动力学相。 在早期重折叠的中间组成中对酰胺的适度保护 观察到由局部序列相互作用形成的两个beta表 5毫秒后重折叠后。 本地保护范围 分子在牵引动力学相中更慢（k大约 2S-1，K小于0.01s-1）。 保护因素由 重折叠100毫秒后改变高pH标记脉冲。 小的 或不存在本地或展开的蛋白质；相反，大多数分子是 在一个或多个部分折叠状态中。 中间状态有 对beta表中残留物的谦虚但很重要的保护 （保护因子10-60），几乎没有α- 螺旋（保护因素小于10）。 标签的模式是 与beta转弯和β发作的角色一致 早期中级。 最近，已经开发了一种化学裂解方法 基于半胱氨​​酸的试剂（EPD-FE）可以连接到蛋白质 链。 抗坏血酸的添加会在 铁中心，将多肽主链分散并裂解 靠近半胱氨酸附着位点的区域可访问残留物 溶剂。 观察到的裂解位点可以用氨基酸映射 测序。 裂解取决于蛋白质构象。 我们 提出的表征了阿疗蛋白的熔融球状态和 使用这种新开发的葡萄球菌核酸酶片段结构 化学裂解技术。 我们将准备许多半胱氨酸 这些蛋白质的变体并表征了裂解模式 在天然和熔融球状状态下观察到。