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.

虽然小蛋白质可能可以在体外自发重折叠， 折叠途径的识别、检测和结构 折叠中间体的表征一直很困难。 最近，注意力转向熔球状态和其他 被认为是模型的蛋白质的非天然平衡状态 用于蛋白质折叠的动力学中间体。 的碎片 已生产出葡萄球菌核酸酶，其也具有以下特性 类似于熔球，即有点致密 具有一些二级结构但没有定义的三级结构 结构。 再折叠过程中的脉冲氢-氘交换已被用于 探讨主链酰胺氢免受溶剂交换的影响 在许多蛋白质的重折叠过程中以及最近 葡萄球菌核酸酶 Pro 117 产生 Gly 变体。 范围 通过二维质子 NMR 测定 39 个残基的交换 重新折叠5毫秒至10秒后。 推断出三个动力学阶段。 早期重折叠中间体中酰胺的适度保护 观察到由局部序列相互作用形成的两个β-折叠 5毫秒重折叠期后。 全程原生保护级别 分子在两个动力学阶段（k 大约为 2s-1，k小于0.01s-1）。 保护系数由下式确定 重折叠 100 ms 后改变高 pH 标记脉冲。 小的 或者不存在天然或未折叠的蛋白质；相反，大多数分子是 处于一种或多种部分折叠状态。 中间状态有 对β-折叠中残留物的适度但重要的保护 （保护系数10-60），并且在α-中几乎没有保护 螺旋（保护因子小于 10）。 标签的样式是 与 β 转角和 β-发夹在形成中的作用一致 的早期中期。 最近开发了一种化学裂解方法，其中 EDT-Fe 基于试剂 (EPD-Fe) 可以通过半胱氨酸侧附着到蛋白质上 链。 添加抗坏血酸会在 铁中心，其在a中扩散和切割多肽主链 靠近残基处的半胱氨酸附着位点的区域 溶剂。 观察到的切割位点可以通过氨基酸来绘制 测序。 切割取决于蛋白质构象。 我们 提出表征脱辅基肌红蛋白的熔球状态和 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。 我们会准备一些半胱氨酸 这些蛋白质的变体并表征切割模式 在自然状态和熔球状态下观察到。