High resolution NMR spectroscopy of nanocrystalline proteins at ultra-high magnetic field
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- Sperling, L.J., Nieuwkoop, A.J., Lipton, A.S. et al. J Biomol NMR (2010) 46: 149. doi:10.1007/s10858-009-9389-9
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Magic-angle spinning (MAS) solid-state NMR (SSNMR) spectroscopy of uniformly-13C,15N labeled protein samples provides insight into atomic-resolution chemistry and structure. Data collection efficiency has advanced remarkably in the last decade; however, the study of larger proteins is still challenged by relatively low resolution in comparison to solution NMR. In this study, we present a systematic analysis of SSNMR protein spectra acquired at 11.7, 17.6 and 21.1 Tesla (1H frequencies of 500, 750, and 900 MHz). For two protein systems—GB1, a 6 kDa nanocrystalline protein and DsbA, a 21 kDa nanocrystalline protein—line narrowing is demonstrated in all spectral regions with increasing field. Resolution enhancement is greatest in the aliphatic region, including methine, methylene and methyl sites. The resolution for GB1 increases markedly as a function of field, and for DsbA, resolution in the C–C region increases by 42%, according to the number of peaks that can be uniquely picked and integrated in the 900 MHz spectra when compared to the 500 MHz spectra. Additionally, chemical exchange is uniquely observed in the highest field spectra for at least two isoleucine Cδ1 sites in DsbA. These results further illustrate the benefits of high-field MAS SSNMR spectroscopy for protein structural studies.
KeywordsNanocrystalline proteinsSolid-state NMR spectroscopySpectral resolutionUltra-high magnetic field
Solid-state NMR (SSNMR) spectroscopy is a powerful tool for studying protein structure and function, uniquely able to address macroscopically disordered proteins. Insights from SSNMR include atomic-resolution structure, site-specific dynamics, metal center chemistry, and orientation of membrane proteins in bilayers (Smith and van Eck 1999; Baldus 2002; McDermott 2004; Opella and Marassi 2004). In recent years, methods have increasingly emphasized the use of uniformly-13C,15N-labeled samples to obtain site-specific information throughout an entire protein in each experiment, a capability for which high magnetic field has been demonstrated to be critical (McDermott et al. 2000; Pauli et al. 2001; Castellani et al. 2002; Bockmann et al. 2003; Igumenova et al. 2004a, b). In principle, this approach greatly enhances throughput and precision of analysis for large peptides and proteins; in practice, however, spectra may be insufficiently resolved to permit resonance assignments and subsequent analysis. To enhance resolution, investigators have made advances in isotopic labeling schemes (LeMaster and Kushlan 1996; Hong and Jakes 1999), decoupling sequences (Bennett et al. 1995; Fung et al. 2000; Detken et al. 2002), and probe designs (Stringer et al. 2005; Doty et al. 2006; Dillmann et al. 2007).
Here we demonstrate systematically the benefits of using ultra-high magnetic fields for magic-angle spinning (MAS) spectra of proteins as nanocrystalline protein preparations. We demonstrate the experimental spectral resolution enhancement at ultra-high field with direct comparison of two proteins over a large range of resonance frequencies, including the highest magnetic field currently available for protein MAS studies (900 MHz). We observe a linear resolution benefit and find that chemical exchange is most prominent at ultra-high field.
Materials and methods
DsbA has been successfully expressed in 100 mg quantities using an expression method described by Marley et al. (2001) E. coli containing DsbA expression plasmid were grown in unlabeled rich medium (LB) to generate large cell mass and then transferred to one-fourth volume of isotopically labeled minimal media prior to induction with IPTG. The cells were lysed by osmotic shock to release the periplasmic proteins. The procedure is intended to wash the cells with sucrose and then lyse the outer membrane with water, but we found that collecting the wash and the water supernatants increased the yield. DsbA was then purified by anion exchange chromatography, and ultimately oxidized with 5 mM glutathione and crystallized in a solution of 30% PEG 8000, 50 mM cacodylate pH 6.5, and 1.5% 2-methyl-2,4-pentanediol. The nanocrystalline solid was packed into limited speed 3.2-mm rotors. The preparation for GB1 has been previously reported (Franks et al. 2005).
Spectra were acquired at B0 fields of 11.7, 17.6, and 21.1 Tesla on a 500 MHz (1H frequency) Varian Infinity Plus spectrometer, a 750 MHz (1H frequency) Varian Unity Inova, and a 900 MHz (1H frequency) Varian Unity Inova. MAS rates were 11.111 kHz at 500 MHz, 12.500 kHz at 750 MHz, and 13.333 kHz at 900 MHz. The 500 and 750 MHz data were acquired with Varian Balun™ 1H–13C–15N 3.2 mm probes, and the 900 MHz data were acquired with a Varian BioMAS™ 1H–13C–15N 3.2 mm probe in double resonance (1H–13C) mode (Stringer et al. 2005). The DsbA data were acquired on a 3.2 mm standard wall rotor with ~6 mg protein on the 750 MHz spectrometer, and on a 3.2 mm limited speed rotor with ~18 mg protein on the 500 and 900 MHz spectrometers. The GB1 data were acquired on three different 3.2 mm standard wall rotors with ~24 mg protein on the 750 and ~10 mg protein on the 500 MHz, and ~12 mg protein on the 900 MHz spectrometers. All pulse sequences utilized tangent ramped cross polarization (Hediger et al. 1994) with TPPM decoupling (Bennett et al. 1995) applied during the acquisition and evolution periods. The variable temperature gas was maintained at −10°C for DsbA with 100 scfh flow. The temperatures for GB1 varied at each field such that GB1 was maintained at 0°C for the 500 MHz experiments and −10°C for the 750 MHz experiments, and the 900 MHz experiments. Chemical shifts were referenced externally with adamantane (Morcombe and Zilm 2003). Data were processed with nmrPipe (Delaglio et al. 1995) with back linear prediction and polynomial baseline (frequency domain) correction applied to the direct dimension. Zero filling to 16,384 in the direct dimension and 8,192 in the indirect dimension was applied and Lorentzian-to-Gaussian apodization was employed for each dimension before Fourier transformation; further details with specifics for each spectrum are found in the respective figure captions.
Results and discussion
To minimize variations arising from technical details such as spectrometer electronics, probe design and sample preparations, we employed highly homologous instrument configurations and standard pulse sequences, applied to replica samples throughout this study. We prepared two proteins: (1) the 56 residue, 6 kDa immunoglobulin binding domain B1 of protein G, GB1 (Gronenborn et al. 1991; Franks et al. 2005) and (2) the 21 kDa soluble disulfide bond forming enzyme, DsbA. Each sample was packed in a limited speed 3.2-mm MAS rotor (Varian, Inc., Palo Alto, CA and Fort Collins, CO). Spectra were collected on three similar spectrometers and triple resonance 1H–13C–15N probes (500 MHz Infinity Plus and 750 MHz Inova with Balun™ 1H–13C–15N probe at the University of Illinois at Urbana-Champaign; 900 MHz Inova with BioMAS™ at the Pacific Northwest National Laboratories, Richland, WA), employing standard pulse sequences, including 1H–13C CP-MAS and two-dimensional (2D) 13C–13C with DARR mixing (Takegoshi et al. 2001; Morcombe et al. 2004) and carefully adjusted TPPM decoupling (Bennett et al. 1995). One can envision further improvements upon application of more recently developed pulse sequences.
Table of chemical shifts for Ile 102 and 113 observed in the 900 MHz (1H frequency) data acquired on U–13C,15N labeled DsbA
Chemical shift (ppm)
In conclusion, our experimental results illustrate that systematic and substantial resolution benefits are observed at ultra-high magnetic field. Here we evaluated these effects directly by comparison of practically identical samples at three magnetic fields, where most relevant hardware performance issues were highly similar. The improved resolution at ultra-high field promises to accelerate interpretation of MAS SSNMR data on large, macroscopically disordered proteins of broad interest, when used in combination with 3D experiments to establish 15N correlations along the peptide backbone.
The authors thank the National Institute of Heath for funding through NIGMS (GM073770), NIGMS/Roadmap Initiative (GM075937) and Molecular Biophysics Training Grant (to LJS and AJN), David Hoyt, Jesse Sears, and Paul Ellis at the Environmental Molecular Science Laboratory (a national scientific user facility sponsored by the Department of Energy Office of Biological and Environmental Research) located at Pacific Northwest National Laboratory and operated for DOE by Batelle for their assistance in acquiring the 900 MHz data, Dr. Donghua Zhou for pulse sequence code, Dr. Trent Franks and Benjamin Fisher of the VOICE NMR Facility for technical assistance.