Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR.
Detection of (15)N in multidimensional NMR experiments of proteins has sparsely been utilized because of the low gyromagnetic ratio (γ) of nitrogen and the presumed low sensitivity of such experiments. Here we show that selecting the TROSY components of proton-attached (15)N nuclei (TROSY (15)NH) yields high quality spectra in high field magnets (>600 MHz) by taking advantage of the slow (15)N transverse relaxation and compensating for the inherently low (15)N sensitivity. The (15)N TROSY transverse relaxation rates increase modestly with molecular weight but the TROSY gain in peak heights depends strongly on the magnetic field strength. Theoretical simulations predict that the narrowest line width for the TROSY (15)NH component can be obtained at 900 MHz, but sensitivity reaches its maximum around 1.2 GHz. Based on these considerations, a (15)N-detected 2D (1)H-(15)N TROSY-HSQC ((15)N-detected TROSY-HSQC) experiment was developed and high-quality 2D spectra were recorded at 800 MHz in 2 h for 1 mM maltose-binding protein at 278 K (τc ~ 40 ns). Unlike for (1)H detected TROSY, deuteration is not mandatory to benefit (15)N detected TROSY due to reduced dipolar broadening, which facilitates studies of proteins that cannot be deuterated, especially in cases where production requires eukaryotic expression systems. The option of recording (15)N TROSY of proteins expressed in H2O media also alleviates the problem of incomplete amide proton back exchange, which often hampers the detection of amide groups in the core of large molecular weight proteins that are expressed in D2O culture media and cannot be refolded for amide back exchange. These results illustrate the potential of (15)NH-detected TROSY experiments as a means to exploit the high resolution offered by high field magnets near and above 1 GHz.
Molecular Profiling Research Center for Drug Discovery, National Institute for Advanced Industrial Science and Technology, Tokyo, 135-0063, Japan.|PRESTO, JST, Tokyo, 135-0063, Japan.
Koh
K
Takeuchi
Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA, 02115, USA.
Haribabu
H
Arthanari
Molecular Profiling Research Center for Drug Discovery, National Institute for Advanced Industrial Science and Technology, Tokyo, 135-0063, Japan. shimada@iw-nmr.f.u-tokyo.ac.jp.|Graduate Schools of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. shimada@iw-nmr.f.u-tokyo.ac.jp.
Ichio
I
Shimada
Department of Biochemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, MA, 02115, USA. gerhard_wagner@hms.harvard.edu.
Gerhard
G
Wagner
D003903:Deuterium / Q000737:chemistry; D058619:Maltose-Binding Proteins / Q000737:chemistry*; D009587:Nitrogen Isotopes / Q000737:chemistry; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011485:Protein Binding; D012680:Sensitivity and Specificity; D047392:beta-Cyclodextrins / Q000737:chemistry
D058619:Maltose-Binding Proteins; D009587:Nitrogen Isotopes; D047392:beta-Cyclodextrins; D003903:Deuterium
Amide back exchange; Deuteration; High field magnet; Nitrogen detection; Protein NMR; TROSY