STUDIES OF PROTEIN FOLDING

与蛋白质相关的合作现象的机制 人们对折叠还没有很好的理解。理解这样的现象是 精确地设计一种功能蛋白质所必需的。因此，我们的研究 旨在了解这些机制。之前的研究已经 允许我们在细胞色素结构中指定4个核心域。一个核心 结构域是包含疏水核心的结构区域，并且 周围的壳作为一个整体可逆地展开。核心域1 可自行折叠，主要由氨基和羧基末端组成 螺旋和亚铁血红素(顶部区域)。核心域2、3和4 分别根据位于左侧的核心( Fe-S键)和血红素的右侧和底部。装配 核心区1、2和3形成疏水性核的主要部分 围绕着亚铁血红素。要知道突变在这方面的影响 酵母iso-2细胞色素c中疏水核心的主要部分 自我传播，四个核心残基I20，M64，L85和M98，一个表面 残基L9和L4的外部残基是由在 ISO-2与马赛特的同源性。C在前几年。观察到的 当前和前几年的情况是：1)即使是I20V这样的天然突变 依赖于核心中的其他残基，在功能上可能是有害的。 这种有害突变效应的上下文依赖性是 这让人想起了细胞色素T的协变量的概念。C)演变；2) 野生型主链的几乎所有质子核磁共振波谱 ISO-2被指定为。M64L、M98L和M64L/M98L突变体的结构 ISO-2与野生型相近。周围的构象迁移率 平均结构按野生型、M98L或M64L的顺序增加， M64L/M88L；3)小幅度、非加性突变效应 在L9I和M64L之间(长距离)和在I20V和M98L之间(短距离 范围)。因此，我们认为上述4个基因突变的影响 核心残基和表面残基会自我传播，这样的 传播是一种重建的过程的表现 在适当折叠的结构区域中的最低自由能状态为 单个单元，并且该结构区域包含由 核心域1、2和3的组装。令人惊讶的是，一个 从iso-2到Horse，大部分残留物在岩心的主要部分 网络电话公司。C导致稳定性没有增加，尽管 野马细胞的稳定性。C优于wt iso-2。以前的研究 建议必须将核心域1、2和3组合在一起才能生成 一种稳定性差异。因此，为了获得关于这一现象起源的线索 现象，2种嵌合的中粒细胞。C已做好准备：一 含有iso-2核心和酵母iso-1细胞。C外壳外加4个额外的NH2 -仅存在于iso-2和另一个iso-1核心中的末端残基 和ISO-2壳层。C102Aiso-1嵌合体的热力学分析 而野生型iso-2表明可能存在独特的核壳 调节细胞色素T稳定性的相互作用。c.

The mechanisms of the cooperative phenomena associated with the protein folding is not well understood. Understanding such phenomena is necessary to precisely design a functional protein. Thus, our studies are aimed at understanding these mechanisms. Previous studies have allowed us to assign 4 core domains in the cytochrome structure. A core domain is a structural region containing a hydrophobic core and surrounding shell which reversibly unfolds as a unit. Core domain 1 folds by itself and consists essentially of the amino and COOH-terminal helices and the heme (the top region). Core domains 2, 3 and 4 respectively, are assigned based on the core located at the left (the Fe -S bond) and right sides and at the bottom of the heme. Assembly of core domains 1, 2 and 3 forms the major part of the hydrophobic core surrounding the heme. To know whether the impact of mutation in this major part of the hydrophobic core in yeast iso-2 cytochrome c propagates itself, four core residues I20, M64, L85 and M98, one surface residues L9 and 4 exterior residues were mutated from residues found in iso-2 to those in horse cyt. c in previous years. The observations of current and previous years are: 1) Even a native mutation such as I20V can be deleterious in function depending on other residues in the core. Such a context dependency of deleterious mutational effect is reminiscent of the concept of the covarion of cyt. c evolution; 2) Almost all proton NMR resonances of the backbone chain of wild type iso-2 are assigned. The structures of M64L, M98L or M64L/M98L mutant iso-2 are close to that of wild type. Conformational mobility around the average structure increases in the order of wild type, M98L or M64L, M64L/M88L; 3) Small magnitude, non-additive mutational effects exist between L9I and M64L (long range) and between I20V and M98L (short range). Thus, we propose that the impact of mutation in the above 4 core and 1 surface residues propagates itself and that such a propagation is a manifestation of a process of re-establishment of the state of lowest free energy in a properly folded structural region as a single unit and that this structural region contains those formed by assembly of core domains 1,2 and 3. Surprisingly, transformation of a majority of residues in the major part of the core from iso-2 to horse cyt. c resulted in no increase in stability despite the much greater stability of wt horse cyt. c over that of wt iso-2. Previous studies suggest that core domains 1, 2 and 3 must be assembled to generate such a stability difference. Thus, to obtain a clue to the origin of this phenomenon, 2 types of chimeric cyts. c have been prepared: one contains iso-2 core and yeast iso-1 cyt. c shell plus 4 extra NH2 -terminal residues which exist only in iso-2 and the other, iso-1 core and iso-2 shell. Thermodynamic analysis of these chimera, C102A iso-1 and wild type iso-2 suggest that there could be unique core-shell interactions which modulates stability of cyt. c.