STUDIES OF PROTEIN FOLDING

我们早期的研究表明， RNA酶A的残基不成比例地使蛋白质不稳定。 作为 结果，大多数核糖核酸酶A衍生物缺乏 4个COOH末端残基不能产生天然的 还原和再氧化后的二硫键。 因此，我们有 假设一些全球性的或长距离的合作互动， 其机制被4 COOH末端的去除所破坏 残基在天然天然蛋白质中起作用，以稳定天然蛋白质。 S-S债券。 这样的互动可能是理解中缺失的部分 的机制，使天然核糖核酸酶A承担最低的自由 能量是由C. B.安芬森 我们随后的研究 葡萄球菌核酸酶已经表明，这种长距离 合作互动可能是普遍的，可能涉及一些独特的 机制 此外，我们最近对细胞色素c的研究 这表明，这种相互作用可能是有效的疏水 核心和相关的核心域域的相互作用， 假设是合作折叠的基础。 核心领域是 定义为含有疏水核的结构区域， 包围着作为一个单元折叠和展开的外壳。 四个核心 在细胞色素C结构中已经指定了结构域。 从而 映射参与这种相互作用的残基，并深入了解 我们对酵母iso-2进行了定点突变， 细胞质c（携带iso-2基因的质粒和酵母菌株GM 3C 2， 来自B.T.的礼物Nall）。 根据先前的研究， 位置20、64、85和98处的表面残基和位置9处的表面残基， 选择了 此外，在位置13、22、90和99处的外部残基 也发生了变异 这些突变（总共16个）是以一种 从iso-2到马的cyt C. 尽管有这样的天然突变， 功能的丧失、恢复、丧失和恢复发生在 单I20 V到双I20 V/M98 L到四倍 I20 V/M98 L/L9 I/M64 L和五元组I20 V/M98 L/L9 I/M64 L/L 85 I 突变 对这些观察结果的分析表明， 位点-位点影响网络，涉及位置9，20，64， 85和98，调节功能。 此外，突变效应 蛋白质的热稳定性，基于695 nm 吸收带和在287 nm处的吸光度，显示非 可加性 对数据的分析表明存在一个网络 的相互作用，涉及残基在位置9，20，64和98。 在这些连接模式中发现了类似的元素 两个网络。 因此，我们假设这两个网络可能是两个 相同的远程合作互动的不同方面。 基于这一假设，这种相互作用可能是导致 限制允许（功能）的场地-场地影响 突变，因此，可能在进化过程中发挥了作用。 真核细胞色素c 为了理解部分 展开状态的iso-2，核磁共振研究正在进行中， 协同工作。

Our earlier studies have shown that removal of 4 carboxy-terminal residues of RNase A disproportionately destabilizes the protein. As a result, a majority of the population of the RNase A derivative lacking the 4 COOH-terminal residues fails to generate the native set of disulfide bonds after reduction and reoxidation. Thus, we have hypothesized that some global or long-range cooperative interactions, whose mechanism is disrupted by removal of the 4 COOH-terminal residues, are operative in the native native protein to stabilize the S-S bonds. Such interactions might be a missing part in understanding of the mechanism which allows native RNase A to assume the lowest free energy as postulated by C.B. Anfinsen. Our subsequent studies of staphylococcal nuclease have suggested that such long-range cooperative interactions may be general and may involve some unique mechanism. Furthermore, our more recent studies of cytochrome c have suggested that such interactions may be operative in the hydrophobic core and related to the core domain-domain interactions which are assumed to be a basis of the cooperative folding. The core domain is defined as a structural region containing a hydrophobic core and the surrounding shell which folds and unfolds as a unit. Four core domains have been assigned in the cytochrome c structure. Thus, to map residues involved in such interactions and obtain insight into the mechanism we have carried out site-directed mutagenesis of yeast iso-2 cyt. c (a plasmid bearing the iso-2 gene and a yeast strain GM3C2 are gifts from B.T. Nall). Based on the previous studies core residues at positions 20, 64, 85 and 98 and a surface residue at position 9 were chosen. In addition, exterior residues at position 13, 22, 90 and 99 were also mutated. The mutations (16 in total) were done in a manner going from iso-2 to horse cyt. c. Despite such native mutations, loss, regain, loss and regain of function occurred going from the single I20V to the double I20V/M98L to the quadruple I20V/M98L/L9I/M64L and to the quintuple I20V/M98L/L9I/M64L/L85I mutation. Analysis of these observations suggests the existence of a site-site influence network involving residues at positions 9, 20, 64, 85 and 98 that modulates function. Furthermore, the mutational effect on heat stability of the protein, determined based on the 695 nm absorption band and the absorbance at 287 nm, has shown non- additivity. Analysis of the data suggests the existence of a network of interactions involving residues at positions 9, 20, 64 and 98. There are found similar elements in the connection pattern in these two networks. Thus, we hypothesize that these two networks may be two different aspects of the same long-range cooperative interactions. Based on this hypothesis such interactions may be responsible for the site-site influence which would restrict allowed (functional) mutations and, therefore, may have played a role in evolution of eukaryotic cytochrome c. To understand the nature of the partially unfolded state of the iso-2, NMR studies are being carried out in the collaborative work.