Insulin is of central importance in human health and provides a general model for studies of protein structure and recognition. How does the hormone bind to the insulin receptor? How does its three-dimensional structure change in the hormone-receptor complex? Of both basic and applied importance, these questions will be investigated by synthetic, biochemical and structural approaches. The following Specific Aims are proposed:
Aim 1 -Toward the Receptor-Active Structure of Insulin: Multidimensional NMR Studies of Paradoxical Analogs. Crystal structures of insulin are now appreciated to represent inactive conformers of the hormone. To deduce the receptor-active structure, we will synthesize active analogs with predicted non-native structures. Of particular interest will be comparison of analogs containing corresponding D and L amino-acid substitutions. Solutions structures will be determined by NMR.
Aim 2 -Mapping the Insulin Receptor by Novel Photo-Crosslinking Analogs. Contact points between insulin and the receptor will be determined by synthesis of A- and B-chain analogs containing a photoactivatable crosslinking amino acid (L-para-azido-Phe). Sites of covalent attachment in the receptor will be mapped by protease digestion and direct micro sequencing.
Aim 3 -Mapping the Insulin-like Growth Factor I (IGF-I) Receptor. Determination of contact points between this growth factor and its receptor will be pursued by the procedures outlined in Aim 2 above, using as parent compound an IGF-I analog consisting of a two-chain molecule embodying the A domain of IGF-I and the B domain of insulin or IGF-I. The feasibility of our Aims are in each case demonstrated by preliminary results. This competing application thus offers the exciting possibility of integrating nonstandard peptide chemistry with biochemistry and biophysics to investigate fundamental aspects of insulin action.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK056673-02
Application #
6350739
Study Section
Metabolism Study Section (MET)
Program Officer
Blondel, Olivier
Project Start
2000-02-01
Project End
2004-01-31
Budget Start
2001-02-01
Budget End
2002-01-31
Support Year
2
Fiscal Year
2001
Total Cost
$278,350
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Pharmacology
Type
Schools of Medicine
DUNS #
114400633
City
New York
State
NY
Country
United States
Zip Code
10029
Smith, Brian J; Huang, Kun; Kong, Geoffrey et al. (2010) Structural resolution of a tandem hormone-binding element in the insulin receptor and its implications for design of peptide agonists. Proc Natl Acad Sci U S A 107:6771-6
Xu, Bin; Huang, Kun; Chu, Ying-Chi et al. (2009) Decoding the cryptic active conformation of a protein by synthetic photoscanning: insulin inserts a detachable arm between receptor domains. J Biol Chem 284:14597-608
Liu, Ming; Wan, Zhu-Li; Chu, Ying-Chi et al. (2009) Crystal structure of a ""nonfoldable"" insulin: impaired folding efficiency despite native activity. J Biol Chem 284:35259-72
Huang, Kun; Chan, Shu Jin; Hua, Qing-xin et al. (2007) The A-chain of insulin contacts the insert domain of the insulin receptor. Photo-cross-linking and mutagenesis of a diabetes-related crevice. J Biol Chem 282:35337-49
Nakagawa, Satoe H; Hua, Qing-xin; Hu, Shi-Quan et al. (2006) Chiral mutagenesis of insulin. Contribution of the B20-B23 beta-turn to activity and stability. J Biol Chem 281:22386-96
Wan, Zhu-li; Huang, Kun; Xu, Bin et al. (2005) Diabetes-associated mutations in human insulin: crystal structure and photo-cross-linking studies of a-chain variant insulin Wakayama. Biochemistry 44:5000-16
Nakagawa, Satoe H; Zhao, Ming; Hua, Qing-xin et al. (2005) Chiral mutagenesis of insulin. Foldability and function are inversely regulated by a stereospecific switch in the B chain. Biochemistry 44:4984-99
Xu, Bin; Hu, Shi-Quan; Chu, Ying-Chi et al. (2004) Diabetes-associated mutations in insulin identify invariant receptor contacts. Diabetes 53:1599-602
Huang, Kun; Xu, Bin; Hu, Shi-Quan et al. (2004) How insulin binds: the B-chain alpha-helix contacts the L1 beta-helix of the insulin receptor. J Mol Biol 341:529-50
Wan, Zhuli; Xu, Bin; Huang, Kun et al. (2004) Enhancing the activity of insulin at the receptor interface: crystal structure and photo-cross-linking of A8 analogues. Biochemistry 43:16119-33

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