Assigning solid-state NMR spectra of aligned proteins using isotropic chemical shifts.
ABSTRACT 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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ABSTRACT: The amphipathic antimicrobial peptide piscidin 1 was studied in magnetically aligned phospholipid bilayers by oriented-sample solid-state NMR spectroscopy. (31)P NMR and double-resonance (1)H/(15)N NMR experiments performed between 25 °C and 61 °C enabled the lipid headgroups as well as the peptide amide sites to be monitored over a range of temperatures. The α-helical peptide dramatically affects the phase behavior and structure of anionic bilayers but not those of zwitterionic bilayers. Piscidin 1 stabilizes anionic bilayers, which remain well aligned up to 61 °C when piscidin 1 is on the membrane surface. Two-dimensional separated-local-field experiments show that the tilt angle of the peptide is 80 ± 5°, in agreement with previous results on mechanically aligned bilayers. The peptide undergoes fast rotational diffusion about the bilayer normal under these conditions, and these studies demonstrate that magnetically aligned bilayers are well suited for structural studies of amphipathic peptides.Biophysical Journal 09/2011; 101(5):1086-94. · 3.67 Impact Factor
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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 01/2011; 209(2):195-206. · 2.30 Impact Factor
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ABSTRACT: Oriented-sample NMR (OS-NMR) has emerged as a powerful tool for the structure determination of membrane proteins in their physiological environments. However, the traditional spectroscopic assignment method in OS NMR that uses the "shotgun" approach, though effective, is quite labor- and time-consuming as it is based on the preparation of multiple selectively labeled samples. Here we demonstrate that, by using a combination of the spin exchange under mismatched Hartmann-Hahn conditions and a recent sensitivity-enhancement REP-CP sequence, spectroscopic assignment of solid-state NMR spectra of Pf1 coat protein reconstituted in magnetically aligned bicelles can be significantly improved. This method yields a two-dimensional spin-exchanged version of the SAMPI4 spectrum correlating the (15)N chemical shift and (15)N-(1)H dipolar couplings, as well as spin-correlations between the (i, i ± 1) amide sites. Combining the spin-exchanged SAMPI4 spectrum with the original SAMPI4 experiment makes it possible to establish sequential assignments, and this technique is generally applicable to other uniaxially aligned membrane proteins. Inclusion of an (15)N-(15)N correlation spectrum into the assignment process helps establish correlations between the peaks in crowded or ambiguous spectral regions of the spin-exchanged SAMPI4 experiment. Notably, unlike the traditional method, only a uniformly labeled protein sample is required for spectroscopic assignment with perhaps only a few selectively labeled "seed" spectra. Simulations for the magnetization transfer between the dilute spins under mismatched Hartmann Hahn conditions for various B (1) fields have also been performed. The results adequately describe the optimal conditions for establishing the cross peaks, thus eliminating the need for lengthy experimental optimizations.Journal of Biomolecular NMR 09/2012; · 2.85 Impact Factor