Using chemical exchange to assign non-covalent protein complexes in slow exchange with the free state: enhanced resolution and efficient signal editing.
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 (1)H and (15)N 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).
Rodríguez
Juan Carlos
JC
Department of Chemistry and Biochemistry, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0359, USA.
Jennings
Patricia A
PA
Melacini
Giuseppe
G
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012680:Sensitivity and Specificity; D013997:Time Factors
D048868:Adaptor Proteins, Signal Transducing; D046912:Multiprotein Complexes; D017868:Cyclic AMP-Dependent Protein Kinases