STUDIES OF PROTEIN FOLDING

我们早期的研究表明，去除4个羧基末端 核糖核酸酶A的残基不成比例地破坏蛋白质的稳定性。作为一名 结果，大多数人群的核糖核酸酶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 假设一些全球或远程的合作互动， 其机制被4-COOH-末端的去除所扰乱 残基，在天然蛋白质中起作用，以稳定 S-S债券。这种互动可能是理解中缺失的一部分 允许原生核糖核酸酶A承担最低自由态的机制 C·B·安芬森假设的能量。我们后续的研究 葡萄球菌核酸酶表明，这种长距离 合作交互可能是一般性的，可能涉及一些独特的 机制。此外，我们最近对细胞色素c的研究已经 表明这种相互作用可能在疏水性 核心，并与核心域-域交互相关，这些交互是 假定为合作折叠的基础。核心域是 定义为包含疏水核心的结构区域 围绕着贝壳，作为一个整体折叠和展开。四核 在细胞色素c结构中已经指定了结构域。因此，要 绘制这种相互作用中涉及的残基并深入了解 我们对酵母iso-2进行了定点突变的机制 网络电话公司。C(含有iso-2基因的质粒和酵母菌GM3C2是 来自B.T.Nall的礼物)。基于之前的研究，核心残留物在 位置20、、85和98以及位置9的表面残留物是 被选中了。此外，第13、22、90和99位的外部残基 也发生了突变。这些突变(总共16个)是以一种方式完成的 从ISO-2到马赛特。C.尽管有这样的天然突变， 功能的丧失、恢复、丧失和恢复 单I20V到双I20V/M98L到四倍 I20V/M98L/L9I/M64L和五路I20V/M98L/L9I/M64L/L85I 突变。对这些观察结果的分析表明， 涉及9，20，64位残基的现场-现场影响网络， 85和98是调节功能的。此外，突变效应 关于蛋白质的热稳定性，根据695 nm测定 吸收带和在287 nm处的吸光度，已经显示出非 可加性。对数据的分析表明存在一个网络 涉及第9、20、和98位残基的相互作用。 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 同一个远程合作互动的不同方面。 根据这一假设，这种相互作用可能是导致 限制允许的站点-站点影响(功能) 突变，因此，可能在进化中发挥了作用 了解真核细胞色素c.部分的性质 Iso-2的展开状态，核磁共振研究正在进行中 协作工作。

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.