This research proposal describes the nuclear magnetic resonance (NMR) structure determination of the 26 kD C-terminal """"""""fibrinogen-like' domain of the protein tenascin. Tenascin is an extracellular matrix protein expressed during early development and is reexpressed in adults tissues during healing processes and in association with tumor metastasis. Many of tenascin's known activities are localized to the C-terminal domain which displays cell and heparin binding activities. Additionally, tenascin's C-terminal domain shares significant sequence homology to other highly interesting proteins, none of which have been structurally determined. The complete assignment of the recombinant C-terminal domain of tenascin will be made using state-of-the-art heteronuclear (1H,13C,15N) 3D and 4D NMR methods. NOE, J-coupling and hydrogen bonding constraints will be obtained using a variety of heteronuclear NMR techniques. These constraints will be used in conjunction with simulated annealing and constrained molecular dynamics to generate structural models. The backcalculation of NOE intensities will be used to verify and improve these NMR structures. The relaxation parameters T1,T2 and the heteronuclear NOE will be measured and fit to various relaxation models to extract intramolecular motional parameters. This information will be used to define possible regions implicated in cell and heparin binding sites. The detailed atomic resolution structure of tenascin's C-terminal domain should be invaluable in localizing the specific amino-acids critical to binding activities and in guiding future mutagenesis and biological investigations into tenascin role in tumor metastasis and tissue development.
