MECHANISMS OF STABILIZATION OF B HEMEPROTEINS

描述：(改编自申请者摘要)总体目标 这一应用的目的是确定结构决定因素 B型血红素蛋白的稳定性和动力学 一个紧密结合的铁卟啉假体基团。移除 这些蛋白质中的血红素基团会改变，但通常不会 抹杀了整个第三纪的结构和稳定性 多肽基质的结构。由此产生的载脂蛋白 为研究结构提供了一个方便的系统 稳定的“折叠”中间体，既不是天然的，也不是 在生理条件下完全展开。之前的工作由 莱科特博士在《进展回顾》和其他人中描述了这一点， 最引人注目的是鲍德温和莱特集团，他们表示 可以考虑单区凋亡素b5和去肌红蛋白。 “模块化。”一个结构域折叠得很快，在没有的情况下非常稳定 亚铁血红素。另一种更直接地参与了血红素的结合，但 在没有义肢组的情况下，稳定性和组织性较差。 这一模块化假说将通过新的、更详细的核磁共振来检验 大鼠肝脏天然和突变可溶性片段的研究 凋亡素b5和重组抹香鲸去肌红蛋白。五 对具体项目进行了描述。(1)温度响应 凋亡素b5的结构和动力学将通过 异核核磁共振技术。我们将寻求一个精致的描述 确定稳定的N和C的结构有多接近 末端组件类似于全蛋白中的末端组件，并且 当假体基团打开内部血红素结合结构域时 被移除。 (2)将使用定点突变来探测特定的元件 二级结构的。特别是，END的pH依赖性 封顶和三级环境在稳定螺旋1和 6，将分别研究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序列的中间部分来构建 (对应于残基44-71)。之前的核磁共振结果表明 这个区域应该形成一个稳定的模子，类似于在 完整的载脂蛋白。如果这被证明是真的，那么模块 用于细胞色素b5折叠的图片将牢牢确立。这个 然后可以分阶段地将第二个模块添加回来，以调查其 在增强稳定性和形成血红素结合部位方面的作用。 (4)将利用三维核磁共振技术确定主要结构 以及抹香鲸去肌红蛋白在溶液中的动态特征。如中所示 在细胞色素b5的情况下，一个组织良好的核心与更 无序区域和定点突变可用于 探索二级和三级结构的个别区域。 (5)截短版本的无肌红蛋白将使用 哺乳动物基因中央外显子的序列。一种类似的 布鲁诺里使用有限的蛋白质分解方法制造了最低限度的肌球蛋白 在罗马的同事，证明了这一方法的可行性。 同样，我们的想法是看看这个外显子是否编码了 去肌红蛋白。

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.