RUI:Insertion of Protease-Binding Loops into Interleukin-18

该项目的目标是确定一种蛋白质的反应区(环)是否能够在新的蛋白质环境中保持其结构和功能。要检查的特定环是来自丝氨酸蛋白酶抑制剂的弹性酶结合环(AT)。这个10个残基的区域已经成功地插入到IL-1b(IL-1b)中，取代了连接第四和第五个b-折叠的转弯。由于最初的插入是将环路放入b片层的一部分，而不是完全按所需的方向插入，因此将通过盒式突变构建新的杂交种。这个环，即抑制物的反应中心，特别令人感兴趣，因为它的完整结构尚未确定；与其他(非抑制性)丝氨酸家族成员的比较表明，它可能采取灵活的a-螺旋的形式。以大豆胰蛋白酶抑制剂为代表的另一类丝氨酸蛋白酶抑制剂的反应环的结构将与AT的结构进行比较。将通过检测重组蛋白中的抗蛋白酶活性来评估环路的完整性。结构将通过X射线结晶学和核磁共振谱(核磁共振)确定。先前的研究结果表明，至少该环的部分功能在新的蛋白质中保持不变。IL-1B似乎是一种很好的支架，可以在其上插入异源环，从而构建许多有用的重组蛋白。这个项目有可能回答关于蛋白质结构的有用和有趣的问题，特别是蛋白酶抑制剂，并解决蛋白质结构预测的问题。它也非常适合培养本科生的研究能力。

The goal of this project is to determine whether the reactive region (a loop) from one protein can maintain its structure and, therefore its function in a new protein environment. The specific loop to be examined is the elastase-binding loop from the serpin (serine protease inhibitor) al-antitrypsin (AT). This ten-residue region has been successfully inserted into Interleukin-1b (IL-1b), replacing the turn connecting the fourth and fifth b-sheets. Because the original insertion placed the loop into part of a b sheet rather than entirely in the desired turn, new hybrids will be constructed by cassette mutagenesis. This loop, the inhibitor's reactive center, is of particular interest, since its intact structure has not been determined; comparison to other (non- inhibitory) members of the serpin family suggests that it may take on the form of a flexible a-helix. The structure of the reactive loops from a different class of serine-protease inhibitors, typified by soybean trypsin inhibitor, will be compared to that obtained for AT. The integrity of the loop will assessed by assaying for anti-protease activity in the recombinant proteins. Structures will be determined by X-ray crystallography and nuclear magnetic resonance spectroscopy (NMR). Previous results indicate that at least some of the functions of the loop are maintained within the new protein. IL-1B appears to be an excellent scaffold on which to insert heterologous loops, allowing construction of many useful recombinant proteins. %%% This project has the potential to answer useful and interesting questions about the structure of proteins in general and protease inhibitors in particular, and to address the problem of prediction of protein structure. It is also ideally suited for training undergraduates in research.