Using chemical exchange to assign non-covalent protein complexes in slow exchange with the free state: Enhanced resolution and efficient signal editing

Authors: Rodríguez, Juan¹; Jennings, Patricia²; Melacini, Giuseppe³

Source: Journal of Biomolecular NMR, Volume 30, Number 2, October 2004 , pp. 155-161(7)

Publisher: Springer

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Abstract:

The formation of a ligand-protein complex oftentimes results in significant chemical shift changes. These changes may occur not only in the binding pocket but also in distal regions of the protein target. Therefore the reassignment of the backbone resonances in the complex is frequently a time consuming challenge. Here we present a suite of resolution-enhanced N_z-exchange NMR experiments useful for rapidly assigning backbone ¹H and ¹⁵N amide resonances of the ligand-bound form of a protein in slow exchange with its free state. Incorporation of semi-constant time frequency labeling periods into 3D N_z-exchange experiments in combination with the collection of resolution-enhanced 2D N_z-exchange difference spectra leads to a powerful set of tools for analyzing protein-ligand complexes. This allows for both the assignment of the bound state and the rapid assessment of the protein binding interface. The proposed methodology is demonstrated on the complex formed by the dimerization-docking domain of the c-AMP-dependent protein kinase and the tethering domain of the dual-binding A-kinase anchoring protein (AKAP).

Keywords: assignments; chemical exchange; ligand binding; protein complexes

Document Type: Research article

DOI: http://dx.doi.org/10.1023/B:JNMR.0000048857.44219.c3

Affiliations: 1: Department of Chemistry and Biochemistry, 011011011011011011011011University of California San Diego, 9500 Gilman Dr., 01101101101101101101101101192093-0359, 011011011011011011011011011La Jolla, 011011011011011011011011011U.S.A, 2: Department of Chemistry and Biochemistry, 011011011011011011011011University of California San Diego, 9500 Gilman Dr., 01101101101101101101101101192093-0359, 011011011011011011011011011La Jolla, 011011011011011011011011011U.S.A, Email: pajennin@c 3: Departments of Chemistry and Biochemistry, 011011011011011011011011McMaster University, 1280 Main Street West, 011011011011011011011011011Hamilton, ON, 011011011011011011011011011Canada L8S 4M1, Email: melacin@mcmaster.ca

Publication date: 2004-10-01

Using chemical exchange to assign non-covalent protein complexes in slow exchange with the free state: Enhanced resolution and efficient signal editing

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