Simultaneous detection of amide and methyl correlations using a time shared NMR experiment: application to binding epitope mapping
 

Simultaneous detection of amide and methyl correlations using a time shared NMR experiment: application to binding epitope mapping
Peter Würtz, Olli Aitio, Maarit Hellman and Perttu Permi
Journal of Biomolecular NMR; 2007; 39(2) pp 97 - 105

Abstract:

Simultaneous recording of different NMR parameters is an efficient way to reduce the overall experimental time and speed up structural studies of biological macromolecules. This can especially be beneficial in the case of fast NMR-based drug screening applications or for collecting NOE restraints, where prohibitively long data collection time may be required. We have developed a novel pulse sequence element that enables simultaneous detection of amide 15N, 1H and methyl 13C, 1H correlations. The coherence selection for the 15N spins can be obtained using the gradient selected and coherence order selective coherence transfer, whereas the hypercomplex (States) method is simultaneously employed for the 13C coherence selection. Experimental verification of proposed time-shared approach for simultaneous detection amide 15N, 1H and methyl 13C, 1H correlations has been carried out with three proteins, human ubiquitin, SH3 domain of human epidermal growth factor receptor pathway substrate 8-like protein (Eps8L1) and maltose binding protein complex with β-Cyclodextrin. In addition, the proposed methodology was applied for ligand binding site mapping on SH3 domain of Eps8L1, using uniformly 15N and fractionally (10%) 13C labeled sample. Our results show that the proposed time-shared 15N/13C-HSQC affords significant time saving (or improved sensitivity) in establishing 15N, 1H and methyl 13C, 1H correlations, thus making it an attractive building block for 3D and 4D dimensional applications. It is also a very efficient tool in protein ligand interaction studies even when combined with cost-effective labeling scheme with uniform 15N and 10% fractional 13C enrichment.