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Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR.

ABSTRACT: Detection of (15)N in multidimensional NMR experiments of proteins has sparsely been utilized because of the low gyromagnetic ratio (?) 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 (?c ~ 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.

SUBMITTER: Takeuchi K

PROVIDER: S-EPMC4749451 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR.

Takeuchi Koh K Arthanari Haribabu H Shimada Ichio I Wagner Gerhard G

Journal of biomolecular NMR 20151023 4

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 transv ...[more]

PMID: 26497830

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Project description:The discovery of the TROSY effect (Pervushin et al. in Proc Natl Acad Sci USA 94:12366-12371, 1997) for reducing transverse relaxation and line sharpening through selecting pathways in which dipole-dipole and CSA Hamiltonians partially cancel each other had a tremendous impact on solution NMR studies of macromolecules. Together with the methyl TROSY (Tugarinov and Kay in J Biomol NMR 28:165-172, 2004) it enabled structural and functional studies of significantly larger systems. The optimal field strengths for TROSY have been estimated to be on spectrometers operating around 900 MHz (21.14 T) for the 1HN TROSY (Pervushin et al. in Proc Natl Acad Sci USA 94:12366-12371, 1997) while the aromatic 13C (13Caro) TROSY is posited to be optimal at around 600 MHz (14.09 T) (Pervushin et al. in J Am Chem Soc 120:6394-6400, 1998b; Pervushin in Q Rev Biophys 33:161-197, 2000). The initial rational was based on the consideration of where the quadratic B0 field dependences of the TROSY relaxation rates reach a minimum. For sensitivity consideration, however, it is interesting to estimate which field strengths yield the tallest peaks. Recent studies of 15N-detected TROSYs suggested that maximal peak heights are expected at 1.15 GHz (27.01 T) although the slowest relaxation rates or longest transverse relaxation times T2 are indeed expected around 900 MHz (21.14 T) (Takeuchi in J Biomol NMR 63:323-331, 2015; Takeuchi et al. in J Biomol NMR 64:143-151, 2016). This was based on the fact that the heights of Lorentzian lines are proportional to B o3/2 * T2 (Bo). Thus, multiplying the parabolic T2(Bo) dependence with the increasing function of B o3/2 shifts the maxima of peak-height field dependence from the T2 maximum at 900 MHz to higher fields. Moreover, besides shifting the peak height maximum for 15N TROSY, this analysis yields estimates for optimal peak heights for 1HN detected TROSY to 1.5 GHz, and to 900 MHz for 13C-detected 13CaroTROSY as is detailed below. To our knowledge, this aspect of field dependence of TROSY sensitivity has not been in the attention of the NMR community but may affect perspectives of NMR at ultra-high fields.

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Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR.

Publications

Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR.

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