The extreme conformational flexibility of small peptide hormones has hindered the elucidation of their biologically active structures by either spectroscopic or crystallographic methods. Since their membrane-bound receptors are, in most cases, inaccessible and not possible to crystallize, it has been difficult. to experimentally address the question of how these unstructured peptides are recognized with extremely high affinities by their receptors. In fact, at the present time there is no structural information about binding of hormone peptides to any protein. One way to obtain information about the high-affinity binding of flexible peptides by proteins is to study the peptides bound with receptor-like affinities to monoclonal antibodies. By studying a number of antibody-peptide complexes, one might not only make use of the specific information for each particular complex, but also begin to delineate some general rules which govern the interactions of small peptide hormones with proteins. We have a panel of monoclonal antibodies which bind with extremely high affinities to Angiotensin II (AII), the major effector of the renin/angiotensin system. We have, in addition, several monoclonals that bind GnRH, the gonadotropin releasing hormone. Crystals have been grown of the uncomplexed and complexed forms of one of these Fabs, Fab-131, and its structure determined and refined by crystallographic methods. In this project we propose to study the three-dimensional structure of several peptide-hormone/Fab complexes The proposed work includes: 1) Completion of the refinement of the structure of the anti-angiotensin(AII) Fab-131 and of the Fab-131/AII cocrystals; 2)Determination of the structures of bound AII analogs by difference Fourier methods and crystallographic refinement of the structure of the complexes; 3) Analysis of the interactions of the binding site of Fab-131 with AH and AH analogs; 4) Design, synthesis and characterization of new AH analogs with potential high affinity for Fab-131; 5) Determination of the structures of the complexes of AII and AII analogs with Fab-133, Fab-110 and Fab-A25; 6)Crystallization of other Fab fragments complexes to AH and GnRH. The structural information obtained in this project will be used to gain insight about receptor/hormone interactions.
