Assigning solid-state NMR spectra of aligned proteins using isotropic chemical shifts

Department of Chemistry and Biochemistry, University of California, San Diego, 9500 Gilman Drive, 0307 La Jolla, CA 92093-0307, USA.
Journal of Magnetic Resonance (Impact Factor: 2.51). 01/2007; 183(2):329-32. DOI: 10.1016/j.jmr.2006.08.016
Source: PubMed


A method for assigning solid-state NMR spectra of membrane proteins aligned in phospholipid bicelles that makes use of isotropic chemical shift frequencies and assignments is demonstrated. The resonance assignments are based on comparisons of 15N chemical shift differences in spectra obtained from samples with their bilayer normals aligned perpendicular and parallel to the direction of the applied magnetic field.

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    • "Here we demonstrate that the isotropic chemical shifts can be " back calculated " from OS solid-state NMR spectra obtained from samples aligned with their bilayer normals parallel and perpendicular to the magnetic field [24]. Since the isotropic chemical shift frequencies vary from site-to-site in a protein they provide a unique " tag " for each residue. "
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    ABSTRACT: A general method for assigning oriented sample (OS) solid-state NMR spectra of proteins is demonstrated. In principle, this method requires only a single sample of a uniformly ¹⁵N-labeled membrane protein in magnetically aligned bilayers, and a previously assigned isotropic chemical shift spectrum obtained either from solution NMR on micelle or isotropic bicelle samples or from magic angle spinning (MAS) solid-state NMR on unoriented proteoliposomes. The sequential isotropic resonance assignments are transferred to the OS solid-state NMR spectra of aligned samples by correlating signals from the same residue observed in protein-containing bilayers aligned with their normals parallel and perpendicular to the magnetic field. The underlying principle is that the resonances from the same residue have heteronuclear dipolar couplings that differ by exactly a factor of two between parallel and perpendicular alignments. The method is demonstrated on the membrane-bound form of Pf1 coat protein in phospholipid bilayers, whose assignments have been previously made using an earlier generation of methods that relied on the preparation of many selectively labeled (by residue type) samples. The new method provides the correct resonance assignments using only a single uniformly ¹⁵N-labeled sample, two solid-state NMR spectra, and a previously assigned isotropic spectrum. Significantly, this approach is equally applicable to residues in alpha helices, beta sheets, loops, and any other elements of tertiary structure. Moreover, the strategy bridges between OS solid-state NMR of aligned samples and solution NMR or MAS solid-state NMR of unoriented samples. In combination with the development of complementary experimental methods, it provides a step towards unifying these apparently different NMR approaches.
    Journal of Magnetic Resonance 04/2011; 209(2):195-206. DOI:10.1016/j.jmr.2011.01.008 · 2.51 Impact Factor
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    ABSTRACT: Magnetically aligned bicelles are an excellent medium for structure determination of isotopically labeled membrane proteins by solid-state NMR spectroscopy. Bicelles are a mixture of long- and short-chain phospholipids that form bilayers in an aqueous medium and align spontaneously in a high magnetic field, for example that of an NMR spectrometer with a 1H resonance frequency between 400 and 900 MHz. Importantly, membrane proteins have been shown to be fully functional in these fully hydrated, planar bilayers under physiological conditions of pH and temperature. We describe a protocol for preparing stable protein-containing bicelles samples that yield high-resolution solid-state NMR spectra. Depending on the details of the protein and its behavior in the lipids, the time for sample preparation can vary from a few hours to several days.
    Nature Protocol 02/2007; 2(10):2332-8. DOI:10.1038/nprot.2007.329 · 9.67 Impact Factor
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    ABSTRACT: In order to develop triple-resonance solid-state NMR spectroscopy of membrane proteins, we have implemented several different (13)C labeling schemes with the purpose of overcoming the interfering effects of (13)C-(13)C dipole-dipole couplings in stationary samples. The membrane-bound form of the major coat protein of the filamentous bacteriophage Pf1 was used as an example of a well-characterized helical membrane protein. Aligned protein samples randomly enriched to 35% (13)C in all sites and metabolically labeled from bacterial growth on media containing [2-(13)C]-glycerol or [1,3-(13)C]-glycerol enables direct (13)C detection in solid-state NMR experiments without the need for homonuclear (13)C-(13)C dipole-dipole decoupling. The (13)C-detected NMR spectra of Pf1 coat protein show a substantial increase in sensitivity compared to the equivalent (15)N-detected spectra. The isotopic labeling pattern was analyzed for [2-(13)C]-glycerol and [1,3-(13)C]-glycerol as metabolic precursors by solution-state NMR of micelle samples. Polarization inversion spin exchange at the magic angle (PISEMA) and other solid-state NMR experiments work well on 35% random fractionally and metabolically tailored (13)C-labeled samples, in contrast to their failure with conventional 100% uniformly (13)C-labeled samples.
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