MECHANISMS OF STABILIZATION OF B HEMEPROTEINS

B 血红素蛋白的稳定机制

• 批准号: 2193607
• 负责人: JULIETTE T LECOMTE
• 金额: $ 18.89万
• 依托单位: PENNSYLVANIA STATE UNIVERSITY, THE
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1999-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2193607
• 关键词: chemical models; chemical stability; cytochrome b5 reductase; molecular dynamics; myoglobin; nuclear magnetic resonance spectroscopy; protein folding; recombinant proteins; site directed mutagenesis

DESCRIPTION: (Adapted from applicant's abstract) The overall goal of this application is to identify the structural determinants of stability and dynamics in b type heme proteins that normally contain a tightly bound iron- porphyrin prosthetic group. Removal of the heme group from these proteins alters, but does not normally obliterate, the structure and stability of the overall tertiary structure of the polypeptide matrix. The resulting apoproteins provide a convenient system for investigating the structures of stable "folding" intermediates which are neither native nor completely unfolded under physiological conditions. Previous work by Dr. Lecomte, which is described in the Progress Review, and by others, most notably the Baldwin and Wright groups, have indicated that single-domain apocytochrome b5 and apomyoglobin can be considered "modular." One domain folds rapidly and is very stable in the absence of heme. The other is involved more directly in heme binding but is less stable and organized in the absence of the prosthetic group. This modular hypothesis will be tested by new and more detailed NMR studies of native and mutant soluble fragments of rat liver apocytochrome b5 and recombinant sperm whale apomyoglobins. Five specific projects are described. (1) The temperature response of the structure and dynamics of apocytochrome b5 will be examined by heteronuclear NMR techniques. A refined description will be sought to determine how closely the structure of the stable N and C terminal module resembles that in the holoprotein and the extent of unfolding of the internal heme binding domain when the prosthetic group is removed. (2) Site-directed mutagenesis will be used to probe specific elements of secondary structure. In particular, the pH dependence of end capping and the role of tertiary context in stabilizing helices 1 and 6, the N and C terminal regions, respectively, will be studied. (3) A truncated model of the stable module in cytochrome b5 will be constructed by excising the middle of the DNA sequence (corresponding to residues 44-71). Previous NMR results suggest that this region should form a stable module similar to that observed in the intact apoprotein. If this proves to be true, then the modular picture for cytochrome b5 folding will be firmly established. The second module can then be added back in stages to investigate its role in conferring stability and forming the heme binding site. (4) 3D NMR techniques will be used to determine the main structural and dynamic features of sperm whale apomyoglobin in solution. As in the case of apocytochrome b5, a well-organized core coexists with more disordered regions, and site-directed mutagenesis can be used to probe individual regions of secondary and tertiary structure. (5) A truncated version of apomyoglobin will be constructed using the sequence of the central exon in the mammalian gene. A similar minimyoglobin has been made using limited proteolysis by Brunori and coworkers in Rome, demonstrating the feasibility of this approach. Again, the idea is to see if this exon codes for a stable module in apomyoglobin.

描述：（改编自申请人摘要）总体目标 本申请的目的是确定 B型血红素蛋白中通常含有 一个紧密结合的铁卟啉辅基。 移除 这些蛋白质的血红素基团发生改变，但通常不会 抹杀，结构和稳定性的整体三级 多肽基质的结构。 由此产生的脱辅基蛋白 提供了一个方便的系统，用于研究 稳定的“折叠”中间体既不是天然的， 在生理条件下完全展开。之前的工作 Lecomte博士，在《进展评论》和其他人的描述中， 最著名的是鲍德温和赖特集团，已经表明， 可以考虑单结构域脱辅基细胞色素b5和脱辅基肌红蛋白 “模块化。“一个结构域折叠迅速， 血红素。另一个更直接地参与血红素结合， 在缺少辅基的情况下不太稳定和有组织。 这一模块化假设将通过新的和更详细的核磁共振测试 大鼠肝脏天然和突变可溶性片段的研究 脱辅基细胞色素B5和重组抹香鲸脱辅基肌球蛋白。五 具体项目进行了说明。(1)的温度响应 脱辅基细胞色素b5的结构和动力学将由 杂波NMR技术。将寻求精确的描述 来确定稳定的N和C的结构 末端模块类似于全蛋白中的末端模块， 当辅基 被移除。 (2)定点突变将用于探测特定元件 二级结构。 特别是，末端的pH依赖性 加帽和三级环境在稳定螺旋1和 6，分别研究N和C末端区域。 (3)细胞色素b5中稳定模块的截断模型将 通过切除DNA序列的中间来构建 （对应于残基44-71）。 先前的NMR结果表明， 这个区域应该形成一个稳定的模块，类似于 完整的载脂蛋白。 如果这被证明是真的，那么模块化 细胞色素b5折叠的图像将被牢固地建立。 的 第二个模块然后可以被添加回阶段，以调查其 在赋予稳定性和形成血红素结合位点中的作用。 (4)3D NMR技术将用于确定主要结构 抹香鲸脱辅基肌红蛋白在溶液中的动力学特征。 如在 在脱辅基细胞色素b5的情况下，组织良好的核心与更多的 无序区域，并且定点突变可以用于 探测二级和三级结构的各个区域。 (5)将使用以下方法构建截短形式的脱辅基肌红蛋白： 哺乳动物基因的中心外显子序列。类似的 Brunori使用有限的蛋白水解制备了微肌红蛋白， 罗马的同事，证明了这种方法的可行性。 同样，我们的想法是看看这个外显子是否编码一个稳定的模块， 去肌红蛋白