1H,15N,13C-triple resonance NMR of very large systems at 900 MHz.
We provide quantitative signal to noise data and feasibility study at 900 MHz for 1H-15N-13C triple resonance backbone assignment pulse sequences obtained from a medium sized 2H, 13C, 15N labeled protein slowed down in glycerol-water solution to mimic relaxation and spectroscopic properties of a much larger protein system with macromolecular tumbling correlation time of 52 and 80 ns, respectively, at 296 and 283 K (corresponding to molecular weights of 130 and 250 kDa). Comparisons of several different schemes for transferring magnetization from proton to nitrogen and back to proton confirms Yang and Kay's 1999 prediction that avoiding the unfavorable relaxation properties of 1H-15N multiple quantum coherence in the TROSY phase cycle of the final 15N-1H transfer before acquisition is crucial for maximal sensitivity from these very large molecular weight systems. We also show results which confirm some predictions regarding the superiority of TROSY at 900 MHz vs. 800 MHz especially as the molecular weights become very large.
Chung
John
J
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA. chung@scripps.edu
Kroon
Gerard
G
D002247:Carbon Isotopes; D005990:Glycerol / Q000737:chemistry; D006801:Humans; D016169:Lymphocyte Function-Associated Antigen-1 / Q000737:chemistry*; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008970:Molecular Weight; D009038:Motion; D009587:Nitrogen Isotopes; D011522:Protons; D011846:Radio Waves; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014867:Water / Q000737:chemistry
D002247:Carbon Isotopes; D016169:Lymphocyte Function-Associated Antigen-1; D046911:Macromolecular Substances; D009587:Nitrogen Isotopes; D011522:Protons; D014867:Water; D005990:Glycerol