STRUCTURAL ANALYSIS OF PROTEIN FOLDING INTERMEDIATES

蛋白质折叠中间体的结构分析

• 批准号: 6138494
• 负责人: Robert O. Fox
• 金额: $ 24.28万
• 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 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6138494
• 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，注意力已经转向熔融球状态和其他 蛋白质的非天然平衡态被认为是模型 蛋白质折叠的动力学中间体。 的片段 已经产生了葡萄球菌核酸酶， 类似于熔融球，即有点紧凑 具有一些二级结构但没有明确的三级结构的结构 结构 在重折叠过程中的脉冲氢-氘交换已被用于 探索保护骨架酰胺氢免受溶剂交换 在许多蛋白质的重折叠过程中， 葡萄球菌核酸酶Pro 117产生Gly变体。 的程度 通过二维质子NMR测定39个残基的交换 复性后5 ms ~ 10 s。 三个动力学阶段推断。 适度保护酰胺在早期重折叠中间组成 观察到两个由局部序列相互作用形成的β折叠 在5 ms重折叠周期之后。 各地的本地保护水平 分子在两个动力学阶段（k约 2s ~（-1），k <0.01s ~（-1）。 保护系数由以下因素确定： 重折叠100 ms后改变高pH标记脉冲。Little 或者不存在天然或未折叠的蛋白质;相反，大多数分子是 处于一个或多个部分折叠状态。 中间状态有 对β折叠中的残留物的适度但重要的保护 （保护系数10-60），在α- 螺旋（保护系数小于10）。 标签的模式是 与β转角和β发夹在形成中的作用一致 早期的中间体。 最近，已经开发了一种化学裂解方法，其中EDT-Fe 基于EPD-Fe的试剂可以通过半胱氨酸侧连接到蛋白质上 链 抗坏血酸的加入在抗坏血酸的存在下产生羟基自由基。 铁中心，其扩散并切割多肽骨架， 在可接近的残基处靠近半胱氨酸附着位点的区域 溶剂后 观察到的切割位点可以通过氨基酸作图 测序 切割依赖于蛋白质构象。 我们 建议表征脱辅基肌红蛋白的熔融球状态， 葡萄球菌核酸酶片段结构，使用这种新开发的 化学裂解技术 我们将准备一些半胱氨酸 这些蛋白质的变体，并表征切割模式 在自然和熔融球状态下观察到。

项目成果

Directed discovery of bivalent peptide ligands to an SH3 domain.

直接发现 SH3 结构域的二价肽配体。

• DOI: 10.1110/ps.03470504
• 发表时间: 2004
• 期刊: Protein science : a publication of the Protein Society
• 作者: Ferguson,MoniqueR;Fan,Xiuzhen;Mukherjee,Munia;Luo,Jinquan;Khan,Raza;Ferreon,JosephineC;Hilser,VincentJ;Shope,RobertE;Fox,RobertO
• 通讯作者: Fox,RobertO

The crystal structure of Escherichia coli heat shock protein YedU reveals three potential catalytic active sites.

大肠杆菌热休克蛋白 YedU 的晶体结构揭示了三个潜在的催化活性位点。

• DOI: 10.1110/ps.03121403
• 发表时间: 2003
• 期刊: Protein science : a publication of the Protein Society
• 作者: Zhao,Yonghong;Liu,Deqian;Kaluarachchi,WarnaD;Bellamy,HenryD;White,MarkA;Fox,RobertO
• 通讯作者: Fox,RobertO

Stability studies of amino acid substitutions at tyrosine 27 of the staphylococcal nuclease beta-barrel.

葡萄球菌核酸酶 β-桶酪氨酸 27 处氨基酸取代的稳定性研究。

• DOI: 10.1021/bi970876r
• 发表时间: 1997
• 期刊: Biochemistry.
• 作者: Bhat,MG;Ganley,LM;Ledman,DW;Goodman,MA;Fox,RO
• 通讯作者: Fox,RO