STRUCTURE-FUNCTION STUDIES OF 70 KD HEAT SHOCK PROTEINS

我们提出了一系列的结构和机制的研究， 70千道尔顿（kDa）热休克样蛋白的成员 家人 基于这些蛋白质中几种的序列数据， 蛋白质家族。 根据其中几个的序列数据 蛋白质，加上牛网格蛋白的功能描述 未包被的ATP酶（HSC 70），我们提出了一个工作模型：所有70 kDa的热休克蛋白从一个共同的祖先进化而来， 保持高度保守的ATP酶结构域， 专门的底物识别域。 他们都应该 相似的三级结构，都可能催化 使用相同的方法进行某种类型的大分子重排 基本机制。 我们将对该蛋白的代表性蛋白进行研究 其生化功能已被充分理解的家族， 牛HSC 70。 我们将解决x射线晶体结构 它的44 kDa ATP酶核心，并将试图结晶， 解析完整HSC 70的结构。 我们正在克隆， HSC70 mRNA的cDNA序列;我们将尝试 构建该蛋白质的重组过量生产者。 我们将 对HSC 70进行蛋白质修饰（诱变）实验 来解决底物识别的机械问题， 构象变化 第二个代表，我们将在 串联体是牛葡萄糖调节蛋白。

We propose a series of structural and mechanistic studies on members of the 70 kilodalton (kDa) heat-shock-like protein family. Based on sequence data for several of these proteins, protein family. Based on sequence data for several of these proteins, plus delineation of the function of the bovine clathrin uncoating ATPase (HSC70), we propose a working model: all 70 kDa heat shock proteins evolved from a common ancestor, maintaining a highly conserved ATPase domain and developing specialized substrate recognition domains. They all should have similar tertiary structures, and all probably catalyze macromolecular rearrangements of some type using the same fundamental mechanism. We will initiate our studies on the representative of this protein family for which a biochemical function is well-understood, bovine HSC70. We will solve the x-ray crystallographic structure of its 44 kDa ATPase core, and will attempt to crystallize and solve the structure of the intact HSC70. We are cloning and will sequence cDNA for the HSC70 mRNA; we will attempt to construct a recombinant overproducer of the protein. We will pursue protein modification (mutagenesis) experiments on HSC70 to address mechanistic questions of substrate recognition and conformational change. A second representative on which we will pursue similar studies in tandem is the bovine glucose regulated protein.