STRUCTURE AND FUNCTION OF THE SMALL HEAT SHOCK PROTEINS

热休克蛋白（HSPs）是由几乎所有的细胞表达， 高温和许多其他压力的条件下。头狼- 堆积相关小（sm）HSP（15 - 30 kDa）在 真核生物，但它们的结构和功能仍然知之甚少。 最近的体外数据表明，smHSP可能作为一种类型的 分子伴侣，体内研究表明它们可能介导 细胞骨架重排我实验室的一个长期研究目标 阐明smHSP的结构和功能。拟议的研究将 研究在高等植物的细胞质中发现的smHSP（PsHSP 18.1和17.7） 以及来自酿酒酵母的同源smHSP（ScHSP 26）。这些 已知smHSP和其它smHSP在体内形成同源寡聚复合物，但 这些配合物的结构没有明确定义。我们可以常规地 分离mg量的高度纯化的重组PsHSP18.1和17.7， 一种稳定的寡聚体形式，并且已经表明这些蛋白质具有 体外伴侣活性。目的是开发一个四元的 smHSPs结构模型，解析超离心，有限 蛋白水解和化学交联将用于检查纯化的 重组Ps HSP 18.1、17.7和ScHSP 26。我们还将继续 用于PsHSP 18.1的X射线结构测定的结晶试验。 smHSP的进一步性质将通过定点PCR进行测试。 提出的结构和功能结构域的诱变。大会 并测试突变蛋白的体外伴侣活性。在 此外，S.酿酒酵母突变体已被分离，需要 升高的ScHSP 26水平的存活率。以前，没有函数可以 归因于HSP 26，因为缺失或过表达菌株 酵母表现出表型的改变。互补克隆 突变基因产生了一个已经鉴定的编码Uso1p的必需基因， 其与哺乳动物p115同源。这两种蛋白质都参与了 ER-高尔基体途径中的细胞内蛋白质转运。HSP26- 依赖性突变体将促进这类化合物的体内功能测定 HSP26与USO 1相互作用的进一步研究 提出提供深入了解ScHSP 26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.