Nitrogen-detected CAN and CON experiments as alternative experiments for main chain NMR resonance assignments

• Published in: Journal of biomolecular NMR 2010-08, Vol.47 (4), p.271-282
• Main Authors: Takeuchi, Koh, Heffron, Gregory, Sun, Zhen-Yu J, Frueh, Dominique P, Wagner, Gerhard
• Format: Article
• Language: English
• Subjects: Bacterial Proteins - chemistry; Biochemistry; Biological and Medical Physics; Biophysics; CAN; Carbon Isotopes - chemistry; CON; Deuterium; Glutathione Transferase - chemistry; HSQC; N direct detection; Nitrogen; Nitrogen Isotopes - chemistry; Nuclear magnetic resonance (NMR); Nuclear Magnetic Resonance, Biomolecular - methods; Physics; Physics and Astronomy; Proteins - chemistry; Research Design; Resonance assignment; Spectroscopy/Spectrometry
• ISSN: 0925-2738; 1573-5001
• DOI: 10.1007/s10858-010-9430-z

Heteronuclear direct-detection experiments, which utilize the slower relaxation properties of low γ nuclei, such as ¹³C have recently been proposed for sequence-specific assignment and structural analyses of large, unstructured, and/or paramagnetic proteins. Here we present two novel ¹⁵N direct-detection experiments. The CAN experiment sequentially connects amide ¹⁵N resonances using ¹³Cα chemical shift matching, and the CON experiment connects the preceding ¹³C′ nuclei. When starting from the same carbon polarization, the intensities of nitrogen signals detected in the CAN or CON experiments would be expected four times lower than those of carbon resonances observed in the corresponding ¹³C-detecting experiment, NCA-DIPAP or NCO-IPAP (Bermel et al. 2006b; Takeuchi et al. 2008). However, the disadvantage due to the lower γ is counteracted by the slower ¹⁵N transverse relaxation during detection, the possibility for more efficient decoupling in both dimensions, and relaxation optimized properties of the pulse sequences. As a result, the median S/N in the ¹⁵N observe CAN experiment is 16% higher than in the ¹³C observe NCA-DIPAP experiment. In addition, significantly higher sensitivity was observed for those residues that are hard to detect in the NCA-DIPAP experiment, such as Gly, Ser and residues with high-field Cα resonances. Both CAN and CON experiments are able to detect Pro resonances that would not be observed in conventional proton-detected experiments. In addition, those experiments are free from problems of incomplete deuterium-to-proton back exchange in amide positions of perdeuterated proteins expressed in D₂O. Thus, these features and the superior resolution of ¹⁵N-detected experiments provide an attractive alternative for main chain assignments. The experiments are demonstrated with the small model protein GB1 at conditions simulating a 150 kDa protein, and the 52 kDa glutathione S-transferase dimer, GST.