Characterization of ligand-binding domains and the ligand binding site on protein disulphide-isomerase

蛋白质在所有生物体中执行大多数关键的生物活性，并且每种蛋白质的个体生物活性关键地取决于其特定的三维结构。在过去的20年里，细胞生物学中最重要的发现之一是认识到细胞中有一种广泛而复杂的机制，致力于确保新制造的蛋白质折叠并正确组装成其特定和独特的三维结构。这种机制包括确保蛋白质快速正确折叠的“折叠催化剂”和防止错误折叠的“分子伴侣”。这种机制现在已经被很好地定义了--就其组成部分的识别而言--但是我们对这些折叠催化剂和分子伴侣在分子细节上是如何工作的知之甚少。这个提议的目的是增加我们对细胞蛋白质折叠机制的一部分的详细分子水平的理解。蛋白质二硫键异构酶（PDI）是一种折叠催化剂和分子伴侣。它是含有二硫键的蛋白质折叠和组装所必需的。二硫键为蛋白质提供额外的稳定性，并且在几乎所有从细胞分泌或暴露于细胞外表面的蛋白质中发现。由于这组蛋白质包括大多数蛋白质激素和其他细胞间信使、激素受体、消化酶、抗体、凝血蛋白和（在其他物种中）毒液毒素、植物贮藏蛋白等，这是一类非常重要的蛋白质。例如，目前用于治疗的大多数人蛋白质药物（如胰岛素、干扰素、生长激素、抗体片段、凝血因子等）。是二硫键结合的蛋白质。据我们所知，PDI或PDI家族的密切相关成员是所有这些蛋白质正确折叠所必需的。因此，更详细地了解PDI不仅对基础细胞生物学而且对医学，兽医和生物技术应用都具有重要意义。令人惊讶的是，经过近30年的研究，PDI的详细结构在分子水平上尚不清楚，因此我们无法精确地描绘它如何帮助新蛋白质折叠并形成正确的二硫键。似乎有一些困难，挫败了传统的方法使用X射线晶体学。我们和我们的合作者在过去2-3年中所做的初步工作表明，我们现在了解了这些困难的基础，并有可能计划如何逐位确定PDI的结构。我们计划从PDI的“域”开始，这对我们来说是最有趣的，因为我们知道它是PDI的“伴侣”属性的关键域。我们将单独确定该结构域的结构，并与相邻结构域结合，作为确定整个结构的一步。我们将不仅关注静态图像，而且还确定PDI的这些部分的灵活性，以便描绘它们在不同时间尺度上经历的分子运动范围。最后，我们将结合一些小蛋白质和更小的片段（肽）研究PDI的这些结构域，旨在了解PDI和它作用的蛋白质如何相互结合，以及它们如何影响彼此的详细结构和动力学。这将最终让我们从分子角度了解PDI是如何工作的。

Proteins carry out most key biological activities in all organisms, and the individual biological activity of each protein is crucially dependent on its specific 3-dimensional structure. One of the most significant discoveries in cell biology in the past 20 years has been the recognition that there is an extensive and complex machinery in cells devoted to ensuring that newly-made proteins fold up and assemble correctly to their specific and unique 3-dimensional structure. This machinery comprises 'folding catalysts' which ensure that proteins fold rapidly and correctly and 'molecular chaperones' which prevent misfolding. This machinery is now quite well-defined -- in terms of the identification of its component parts -- but we know very little about how these folding catalysts and chaperones work in molecular detail. The aim of this proposal is to increase our detailed molecular-level understanding of one part of the cellular protein folding machinery. Protein disulphide-isomerase (PDI) is a folding catalyst and chaperone which has been known for many years. It is absolutely required for the folding and assembly of proteins that contain disulphide bonds. Disulphide bonds provide proteins with additional stability and are found in almost all proteins which are secreted from cells or exposed at the extracellular surface of cells. Since this group of proteins includes most protein hormones and other intercellular messengers, hormone receptors, digestive enzymes, antibodies, blood clotting proteins, and (in other species) venom toxins, plant storage proteins etc., this is a very significant class of proteins. For example, most of the human protein drugs which are currently used in therapy (such as insulin, interferons, growth hormones, antibody fragments, blood clotting factors etc). are disulphide-bonded proteins. To the best of our knowledge, PDI or a closely related member of the PDI family, is required for the correct folding of all such proteins. Consequently, more detailed understanding of PDI would be significant not only for basic cell biology but also for medical, veterinary and biotechnological applications. Surprisingly, after almost 30 years of study, the detailed structure of PDI is not known at the molecular level, and so we cannot picture precisely how it acts to assist newly-made proteins to fold and form correct disulphide bonds. There appear to be some difficulties which have frustrated conventional approaches using x-ray crystallography. Preliminary work that we and our collaborators have done over the past 2-3 years suggests that we now understand the basis of these difficulties and makes it possible to plan how to determine the structure of PDI bit-by-bit. We plan to start with the 'domain' of PDI which is most interesting to us, because we know that it is the key domain for the 'chaperone' properties of PDI. We will determine the structure of this domain alone and in combination with a neighbouring domain, as a step towards determining the whole structure. We will not focus simply on a static picture but also determine the flexibility of these parts of PDI, in order to picture the range of molecular motions they undergo on various timescales. Finally we will study these domains of PDI in combination with some small proteins and even smaller fragments (peptides) aiming to understand how PDI and the proteins on which it acts bind to each other and how each influences the detailed structure and dynamics of the other. This will finally give us some insight into how PDI works, in molecular terms.