We have probed the binding of a synthetic peptide corresponding to the region 550-585 of the α subunit of dystroglycan with a recombinant protein fragment corresponding to the N-terminal extracellular region of β-dystroglycan (654-750), using NMR in solution. In a 30:1 molar ratio, the peptide binds to the recombinant protein fragment in the fast/intermediate exchange regime. By monitoring the peptide intra-residue HN-Hα peak volumes of the 2D TOCSY NMR spectra, both in the absence and in the presence of the recombinant fragment, we determined the differential binding affinities of each amino acid. We found that the residues in the region 550-565 (SWVQFNSNSQLMYGLP) are more influenced by the presence of the protein, whereas the C-terminal portion is marginally involved. These NMR results have been confirmed by solid-phase binding assays.
|Original language||English (US)|
|Number of pages||5|
|State||Published - Jun 22 2001|
Bibliographical noteFunding Information:
The authors would like to thank A. Mascioni for assistance in purifying the peptide, K. Mayo, R. Di Fonzo and T. Petrucci for their helpful comments, and D. Live and B. Ostrowski for helping with the NMR experiments. NMR instrumentation was provided with funds from the NSF (BIR-961477) and the University of Minnesota Medical School. The financial support of CNR, target project ‘Biotechnology’ and Telethon-Italy (Grant 1267) is gratefully acknowledged.
- Binding epitope
- Mapping binding site
- Nuclear magnetic resonance spectroscopy
- Protein-protein interaction