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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
5P01GM051362-05
Application #
6316668
Study Section
Project Start
2000-06-01
Project End
2002-05-31
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
5
Fiscal Year
2000
Total Cost
$366,194
Indirect Cost
Name
Johns Hopkins University
Department
Type
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Maynard, Ernest L; Berg, Jeremy M (2007) Quantitative analysis of peroxisomal targeting signal type-1 binding to wild-type and pathogenic mutants of Pex5p supports an affinity threshold for peroxisomal protein targeting. J Mol Biol 368:1259-66
Amzel, L Mario; Siebert, Xavier; Armstrong, Anthony et al. (2005) Thermodynamic calculations in biological systems. Biophys Chem 117:239-54
Siebert, Xavier; Amzel, L Mario (2004) Loss of translational entropy in molecular associations. Proteins 54:104-15
Gatto Jr, Gregory J; Maynard, Ernest L; Guerrerio, Anthony L et al. (2003) Correlating structure and affinity for PEX5:PTS1 complexes. Biochemistry 42:1660-6
Kang, Lin-Woo; Gabelli, Sandra B; Bianchet, Mario A et al. (2003) Structure of a coenzyme A pyrophosphatase from Deinococcus radiodurans: a member of the Nudix family. J Bacteriol 185:4110-8
Nezami, Azin; Luque, Irene; Kimura, Tooru et al. (2002) Identification and characterization of allophenylnorstatine-based inhibitors of plasmepsin II, an antimalarial target. Biochemistry 41:2273-80
Luque, Irene; Leavitt, Stephanie A; Freire, Ernesto (2002) The linkage between protein folding and functional cooperativity: two sides of the same coin? Annu Rev Biophys Biomol Struct 31:235-56
Ahmed, Hafiz; Bianchet, Mario A; Amzel, L Mario et al. (2002) Novel carbohydrate specificity of the 16-kDa galectin from Caenorhabditis elegans: binding to blood group precursor oligosaccharides (type 1, type 2, Talpha, and Tbeta) and gangliosides. Glycobiology 12:451-61
Velazquez-Campoy, A; Kiso, Y; Freire, E (2001) The binding energetics of first- and second-generation HIV-1 protease inhibitors: implications for drug design. Arch Biochem Biophys 390:169-75
Velazquez-Campoy, A; Todd, M J; Vega, S et al. (2001) Catalytic efficiency and vitality of HIV-1 proteases from African viral subtypes. Proc Natl Acad Sci U S A 98:6062-7

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