传统认为蛋白质的结构决定其功能，近年来对蛋白质中内在无序区域的研究挑战了这一观点。这些区域被认为是多种动态结构的集合，在转录与翻译调控、细胞信号转导、分子识别等关键过程中发挥重要作用。在分子伴侣蛋白这一大类帮助生物大分子折叠与组装的关键蛋白中也存在内在无序区域，探索其功能成为分子伴侣研究领域的一个热点问题。新型分子伴侣蛋白Spy约30%的残基位于无序区域，前期研究表明增加Spy内在无序性能够增加其分子伴侣活力，但作用机制还有待确定。本项目拟深入探讨Spy内在无序性区域在其识别、结合与释放底物中的作用，并提出Spy不依赖ATP和辅助因子促进底物蛋白折叠的分子机制。本项目将为分子伴侣内在无序区域的功能性研究提供直接的实验证据，揭示新型分子伴侣机制，为解析如Hsp60、Hsp70等复杂分子伴侣系统的机制提供新思路，从而显著提高对蛋白质内在无序、分子伴侣结构与功能的关系和蛋白质折叠机制的理解。

It has long been assumed that protein structure dictates function, however, this notion has been challenged by recent advance on the study of intrinsically disordered regions in proteins. Unstructured but functional, these regions can exist simultaneously in different, yet highly dynamic conformations and play important roles in key biological processes, such as regulation of transcription and translation, cellular signal transduction, and molecular recognition. Molecular chaperones are a large and diverse group of proteins that assist in the folding and assembly of macromolecules. It is becoming clear that structural disorder also exists in molecular chaperones, and its roles in the mechanism of chaperone's action have increasingly been explored in the last few years. The recently discovered chaperone Spy possesses long disordered regions that account for 30% of its residues. Our previous work on enhancing Spy's chaperone activity has showed that the activity-enhancing Spy variants were generally more unstable than is wild type Spy and had increased apparent flexibility, indicating that intrinsic disorder is important for determining Spy's activity. We decide to further investigate the functional contribution of Spy's intrinsically disordered regions, by focusing on their roles in substrate recognition, binding, and release using a combination of bacterial genetic, biochemical and biophysical techniques. We also aim to determine the mechanism of Spy by which it assists the folding/refolding of substrate proteins in an ATP-independent manner. The implementation of our proposed research will provide direct experimental evidence of the role of disorder in chaperone function and may also reveal novel chaperone mechanisms. Our result will also bring new insights in investigating more complex chaperone systems, such as the Hsp60 and Hsp70 chaperone systems, therefore advance our understanding of protein intrinsic disorder, the function of molecular chaperones and the mechanism of protein folding.