The proposed research aims to develop a methodology to isolate high affinity binders to the transmembrane regions of integrin alpha (v) beta (3). Little is known about the binding patterns and preferences of transmembrane helices. Using a tagged target integrin transmembrane sequence selection of a high affinity binder will be isolated from a library of mutants in either a bilayer or micelle. Sequencing of the high affinity binder is afforded through protein sequencing or tandem fractionating MS/MS. Results from library selection are used to refine Monte Carlo parameters to optimize calculations for predicting membrane helix associations. Integrin alpha(v)beta(3) is involved in neovascularization and is found in tumor cells, but absent in normal cells. The high affinity binder and target complex will be investigated by analytical ultracentrifugation (AUC) and fluorescence resonance energy transfer (FRET) to determine the dimerization binding energy. NMR will be used to probe the geometry of interaction and identify the helical interface. Incorporation of high affinity binders into cells line with determine if they can be used as dominant negatives as cellular activity can be followed with monoclonal antibodies.
| Tatko, Chad D; Nanda, Vikas; Lear, James D et al. (2006) Polar networks control oligomeric assembly in membranes. J Am Chem Soc 128:4170-1 |
