Simultaneous detection of amide and methyl correlations using a time shared NMR experiment: application to binding epitope mapping.
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 (15)N, (1)H and methyl (13)C, (1)H correlations. The coherence selection for the (15)N spins can be obtained using the gradient selected and coherence order selective coherence transfer, whereas the hypercomplex (States) method is simultaneously employed for the (13)C coherence selection. Experimental verification of proposed time-shared approach for simultaneous detection amide (15)N, (1)H and methyl (13)C, (1)H 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 beta-Cyclodextrin. In addition, the proposed methodology was applied for ligand binding site mapping on SH3 domain of Eps8L1, using uniformly (15)N and fractionally (10%) (13)C labeled sample. Our results show that the proposed time-shared (15)N/(13)C-HSQC affords significant time saving (or improved sensitivity) in establishing (15)N, (1)H and methyl (13)C, (1)H 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 (15)N and 10% fractional (13)C enrichment.
Würtz
Peter
P
Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory University of Helsinki, P.O. Box 65, Helsinki 00014, Finland.
Aitio
Olli
O
Hellman
Maarit
M
Permi
Perttu
P
D000577:Amides / Q000737:chemistry*; D002247:Carbon Isotopes; D002352:Carrier Proteins / Q000737:chemistry; D018604:Epitope Mapping / Q000379:methods*; D004926:Escherichia coli; D006801:Humans; D058619:Maltose-Binding Proteins; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011522:Protons; D012996:Solutions; D013997:Time Factors; D025801:Ubiquitin / Q000737:chemistry; D018909:src Homology Domains
D000577:Amides; D002247:Carbon Isotopes; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D009587:Nitrogen Isotopes; D011522:Protons; D012996:Solutions; D025801:Ubiquitin
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