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al-抗胰蛋白酶（AT）的弹性酶结合环。这十个残基区已成功插入到白细胞介素-1b （IL-1b）中，取代了连接第4和第5个b-sheet的转位。由于最初的插入将环置于b片的一部分，而不是完全按照期望的方向，因此新的杂交种将通过盒式诱变构建。这个环，抑制剂的反应中心，是特别感兴趣的，因为它的完整结构尚未确定；与其他（非抑制性）蛇形蛋白家族成员的比较表明，它可能采取灵活的a-螺旋的形式。从不同种类的丝氨酸蛋白酶抑制剂，以大豆胰蛋白酶抑制剂为代表，反应环的结构将与获得的AT进行比较。环的完整性将通过检测重组蛋白的抗蛋白酶活性来评估。结构将由x射线晶体学和核磁共振波谱（NMR）确定。先前的结果表明，至少环路的一些功能在新蛋白中得到维持。IL-1B似乎是一个很好的支架，在其上插入异源环，允许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的一般结构，特别是蛋白酶抑制剂的有用和有趣的问题，并解决蛋白质结构的预测问题。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.