The broad, long-term objective of the proposed project is to understand the basis of protein-ligand interactions. The focus is on molecular recognition by surface loops of beta-sandwich proteins, such as found in antibodies (Abs). Although the structure-function relationships of Abs have been an area of intense research activities, questions of fundamental importance remain to be answered; they include conformational changes of the loops upon ligand binding, and the relationships between loop flexibility and affinity. Such questions would be best addressed by solution studies using NMR spectroscopy, but the large size of Abs and the low solubility of small functional fragments have seriously hampered NMR studies of Abs. We propose a novel approach to establish a model system to study the structure and function of surface binding loops using NMR spectroscopy and other biophysical techniques. The small, soluble and stable scaffold of fibronectin type III domain (Fn3) will be used. Fn3 has a structure closely related to Abs.
Our specific aims are: 1) To test whether it is possible to replace Fn3 loops with foreign loops without the loss of the global structure. Antigen binding loops (CDRs) of Ab fragments of known structure will be grafted on Fn3 using structure-based design, and stability of mutant proteins will be characterized. 2) To examine the possibility of raising Fn3-based mini-Abs (FnAbs) which can specifically bind a given ligand, by selecting mutants from combinatorial libraries in which surface loops of Fn3 ;are randomized. The phage display technique will be used, 3) To characterize the solution conformation anal ligand interactions of FnAbs in the free and complexed states by state-of-the-art NMR spectroscopy. The initial focus is on mapping the binding surface of novel complexes and on identifying specific FnAb-ligand contacts. Solution structures of FnAb complexes will be determined. 4) To improve the affinity and specificity of FnAbs by iterative processes of designing and/or screening new interactions followed by structural analysis. Structural information derived from this study will provide new insights into the conformation and dynamics of surface binding loops which are essential elements of many medically important proteins. Designed FnAbs with high affinity and specificity will have immediate use as a building block of recombinant protein reagents for diagnostic and therapeutic applications.
