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.
