The extreme flexibility of small peptide hormones has hindered attempts at elucidating their solution structure. Structural studies of their receptor bound conformations have not been possible either because the receptors (for those that they are known) are complex, integral membrane proteins. Thus, important questions about high affinity recognition of small, flexible peptide hormones have remained elusive. In this project awe are carrying out structural studies on complexes of two peptide hormones -- angiotensin II and gonadotrophin releasing hormone-- with high affinity monoclonal antibodies. These studies are directed at understanding all aspects of high affinity recognition of the hormones including the evaluation of thermodynamical properties from structural data. In addition, the structural information will be used in the design of analogs of the hormones that could bind the antibodies (and may be the natural receptors) with high affinity. As part of this project we have determined the three dimensional structure of a Fab/All complex (Fab-131), measured the thermodynamics of binding, developed methods that predict affinities based on structural information, and identified several members of a combinatorial cyclic peptide library that bind to the antibody. In the next period we propose: 1) to determine and refine the structures of the complexes of Fab 131 with AII analogs --including the cyclic peptides we identified-- and AII antagonists; 2) to design and characterize new AII analogs with potential high affinity for Fab-131; 3) to complete the determination of the structure of the Fab 110-AII complex and the analysis of the energetics of binding. Determination of the deltaG, deltaH, deltaCp, and deltaS of binding; 4) to determine the structure of the complexes of AII and AII analogs with Fab-133, Fab-110 and Fab-A25; 5) to crystallize other Fab fragments complexed to AII and GnRH.
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