Studies of the Folding of Thioredoxin by Fragment Complementation on the Development of New Tools to Improve the Understanding of Biomolecular Structure and Function

[507255]这项研究的目的是确立纸的折叠原理。所选择的方法是研究蛋白质片段家族的结构、稳定性、动力学和折叠。大肠杆菌硫氧还蛋白是一种具有良好特征的混合结构小单体蛋白，本文提出将其分解成片段。它们将使用生物化学、化学和分子生物学的工具生成。这些片段将使用生物化学和生物物理工具的组合来表征，并增加结构细节的水平。圆二色性在远、近紫外，荧光，多维核磁共振波谱和计算机建模也将使用。本提案的教育目的是利用逆预测方法来补充生物化学专业标准的一学期物理化学课程，逆预测方法包括建立一个大分子的物理3d模型，并用它来预测其生物物理性质。将结构变化与生物功能变化联系起来的能力是理解生物大分子结构-功能关系的关键。然而，蛋白质结构的建立速度与新氨基酸序列的产生速度不同。因此，解决“蛋白质折叠问题”，即仅根据蛋白质的一级序列预测蛋白质的三维结构，已成为一个热门研究领域。在理解蛋白质中常见的一种结构-螺旋的形成原理方面已经取得了很大进展。然而，人们对另一种同样重要的结构成分-薄片的形成知之甚少。本研究将比较硫氧还蛋白片段及其结合产物与原蛋白片段的结构、稳定性和折叠，以阐明尚不清楚的-片的折叠。＊＊＊

9507255 Tasayco The goal of the research is to establish the principles underlying the folding of -sheets. The approach chosen is to study the structure, stability, dynamics and folding of a family of protein fragments. It is proposed to dissect E. coli thioredoxin, a well- characterized small monomeric protein with a mixed -structure, into fragments. They will be generated using tools of biochemistry, chemistry and molecular biology. These fragments will be characterized using a combination of biochemical and biophysical tools with increasing level of structural detail. Circular dichroism in the far and near UV, fluorescence, multidimensional nuclear magnetic resonance spectroscopy and computer modeling will also be used. The educational aspect of this proposal aims to complement the lectures of the standard one-semester Physical Chemistry course for Biochemistry majors using the Inverse Prediction Approach, which consists of building a physical 3D-model of a macromolecular and using it to predict its biophysical properties. %%% The ability to correlate structural changes with ahanges in the biological function holds the key to the understanding of the structure-function relationship of biological macromolecules. However, protein structures are not established at the same pace at which new amino acid sequence are generated. Thus solving the "protein folding problem," that is, predicting the three dimensional structure of a protein based solely on its primary sequence, has become an area of intense research. Much progress has been made in the understanding of the principles behind the formation of one kind of structure commonly found in proteins, the -helix. However, little is known about the formation of -sheets, another equally important structural component. This research will compare the structure, stability and folding of the fragments of thioredoxin and their association products with those of the original protein to elucidate the folding of the still little understood -sheets. ***