The objective of this project is to obtain high resolution solution structures of proteins containing the SH (Src-Homology) domains 2 and 3 using multiple dimensional NMR. These domains are found in many proteins that are involved in signal transducing pathways and have been implicated in the inter- and intra-molecular regulation of kinase and other enzyme activities and in the oncogenic potential of these proteins. As well, one SH2 domain has been demonstrated to be sufficient for the high affinity binding of tyrosyl-phosporylated proteins and and another SH3 domain may bind actin filaments of the cellular cytoskeleton. This study is proposed in order to generate the first three dimensional structures of these novel domains and to identify their intramolecular interactions and phosphotyrosyl-containing protein binding sites. The immediate goal is to proceed from circular dichroism and two-dimensional NMR studies already completed with unlabelled protein to a complete assignment and secondary structure analysis of a N15 labelled 107 residue protein containing the 80- amino acid SH2 domain from c-abl protein. A high resolution tertiary structure determination using interproton distance constraints and torsion angles from C-13 and N-15 labeled protein would be generated. Amino acid residues involved in binding phosphotyrosyl containing substrate would be identified by NMR. Since the transforming activity of several oncogenes has been traced to mutations in their SH2 domains, the three dimensional structure of the SH2 domain of the c-abl proto-oncogene should provide a structural explanation for their oncogenic activity, and may suggest rational routes to therapies for related conditions.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM047021-03
Application #
2184499
Study Section
Biophysical Chemistry Study Section (BBCB)
Project Start
1992-02-01
Project End
1995-06-30
Budget Start
1994-02-01
Budget End
1995-06-30
Support Year
3
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Rockefeller University
Department
Chemistry
Type
Other Domestic Higher Education
DUNS #
071037113
City
New York
State
NY
Country
United States
Zip Code
10065
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Xu, Rong; Liu, Dongsheng; Cowburn, David (2012) Abl kinase constructs expressed in bacteria: facilitation of structural and functional studies including segmental labeling by expressed protein ligation. Mol Biosyst 8:1878-85
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Piserchio, Andrea; Cowburn, David; Ghose, Ranajeet (2012) Expression and purification of Src-family kinases for solution NMR studies. Methods Mol Biol 831:111-31
Bhattacharya, Shibani; Zhang, Hongtao; Cowburn, David et al. (2012) Novel structures of self-associating stapled peptides. Biopolymers 97:253-64
Zhang, Hongtao; Curreli, Francesca; Zhang, Xihui et al. (2011) Antiviral activity of ?-helical stapled peptides designed from the HIV-1 capsid dimerization domain. Retrovirology 8:28
Ferrage, Fabien; Reichel, Amy; Battacharya, Shibani et al. (2010) On the measurement of ¹?N-{¹H} nuclear Overhauser effects. 2. Effects of the saturation scheme and water signal suppression. J Magn Reson 207:294-303
Tait, Shirley; Dutta, Kaushik; Cowburn, David et al. (2010) Local control of a disorder-order transition in 4E-BP1 underpins regulation of translation via eIF4E. Proc Natl Acad Sci U S A 107:17627-32

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