Speeding up direct (15)N detection: hCaN 2D NMR experiment.
Experiments detecting low gyromagnetic nuclei have recently been proposed to utilize the relatively slow relaxation properties of these nuclei in comparison to (1)H. Here we present a new type of (15)N direct-detection experiment. Like the previously proposed CaN experiment (Takeuchi et al. in J Biomol NMR 47:271-282, 2010), the hCaN experiment described here sequentially connects amide (15)N resonances, but utilizes the initial high polarization and the faster recovery of the (1)H nucleus to shorten the recycling delay. This allows recording 2D (15)N-detected NMR experiments on proteins within a few hours, while still obtaining superior resolution for (13)C and (15)N, establishing sequential assignments through prolines, and at conditions where amide protons exchange rapidly. The experiments are demonstrated on various biomolecules, including the small globular protein GB1, the 22 kDa HEAT2 domain of eIF4G, and an unstructured polypeptide fragment of NFAT1, which contains many SerPro sequence repeats.
Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Maayan
M
Gal
Katherine A
KA
Edmonds
Alexander G
AG
Milbradt
Koh
K
Takeuchi
Gerhard
G
Wagner
D039603:Eukaryotic Initiation Factor-4G / Q000737:chemistry; D006801:Humans; D050778:NFATC Transcription Factors / Q000737:chemistry; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D018080:Receptors, GABA-B / Q000737:chemistry
C113920:EIF4G1 protein, human; D039603:Eukaryotic Initiation Factor-4G; D050778:NFATC Transcription Factors; C494006:NFATC2 protein, human; D009587:Nitrogen Isotopes; D011506:Proteins; D018080:Receptors, GABA-B