STRUCTURE AND FUNCTION OF THE SMALL HEAT SHOCK PROTEINS

热休克蛋白(HSPs)几乎由所有的细胞表达。 高温和许多其他压力的条件。阿尔法- 与晶体蛋白相关的小(Sm)热休克蛋白(15-30 kDa)普遍存在于 但对它们的结构和功能仍知之甚少。 最近的体外数据表明，smHSPs可能是一种 分子伴侣，体内研究表明它们可能在 细胞骨架重排。我实验室研究的一个长期目标 目的是阐明smHSP的结构和功能。拟议的研究将 高等植物胞质中发现的单链热休克蛋白的研究(PsHSP 18.1和17.7) 和酿酒酵母的同源smHSP(ScHSP26)。这些 和其他smHSP已知在体内形成同源低聚复合体，但 这些复合体的结构并不是很明确。我们可以照例 分离纯化高纯度重组PsHSPl8.1和17.7mg量 一种稳定的寡聚体形式，并表明这些蛋白质具有 体外伴侣活性。以发展第四纪为目标 SMHSPs的结构模型，分析超速离心，有限 蛋白水解法和化学交联法将用于检测纯化的 重组菌株HSP18.1、17.7和ScHSP26。我们也会继续 PsHSP18.1晶体结构测定的晶化实验 SMHSP的进一步性能将通过现场定向测试 建议的结构和功能结构域的突变。该组件 并将在体外测试突变蛋白的伴侣活性。在……里面 此外，还分离到了一株酿酒酵母突变体，需要 ScHSP26水平升高，以提高生存能力。以前，任何函数都不能 归因于HSP26，因为既没有缺失也没有过表达 酵母菌的表型发生了变化。基因的互补克隆 突变基因产生了一个编码Uso1p的已鉴定的必要基因， 它与哺乳动物的p115同源。这两种蛋白质都参与了 内质网-高尔基体途径中的细胞内蛋白运输。这个HSP26- 依赖突变体将促进这一类别的体内功能分析 HSP26与USO1相互作用的进一步研究如下 建议深入了解ScHSP26的功能。

Heat shock proteins (HSPs) are expressed by virtually all cells under conditions of high temperature and many other stresses. The alpha- crystallin-related, small (sm) HSPs (15-30 kDa) are ubiquitous among eukaryotes but their structure and function remain poorly understood. Recent in vitro data indicate that the smHSPs may act as a type of molecular chaperone, and in vivo studies suggest they may mediate cytoskeletal rearrangements. A long-term goal of research in my laboratory is to elucidate smHSP structure and function. The proposed research will study smHSPs found in the cytosol of higher plants (PsHSP 18.1 and 17.7) and the homologous smHSP from Saccharomyces cerevisiae (ScHSP26). These and other smHSPs are known to form homo-oligomeric complexes in vivo, but the structure of these complexes is not well-defined. We can routinely isolate mg quantities of highly purified recombinant PsHSPl8.1 and 17.7 in a stable oligomeric form, and have shown that these proteins have chaperone activity in vitro. With the aim of developing a quaternary structure model of the smHSPs, analytical ultracentrifugation, limited proteolysis, and chemical crosslinking will be used to examine purified recombinant Ps HSP 18.1, 17.7, and ScHSP26. We will also continue crystallization trials for X-ray structure determination of PsHSP18.1. Further properties of the smHSPs will be tested by site-directed mutagenesis of proposed structural and functional domains. The assembly and in vitro chaperone activity of the mutant proteins will be tested. In addition, an S. cerevisiae mutant has been isolated which requires elevated ScHSP26 levels for viability. Previously, no function could be ascribed to HSP26 because neither deletion nor over-expression strains of yeast showed alterations in phenotype. Complementation cloning of the mutant gene yielded an already identified, essential gene encoding Uso1p, which is homologous to mammalian p115. Both proteins are involved in intracellular protein transport in the ER-Golgi pathway. This HSP26- dependent mutant will facilitate in vivo functional assays for this class of HSPs, and further studies of the interaction of HSP26 with USO1 are proposed to provide insight into ScHSP26 function.