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Determination of 15N chemical shift anisotropy from a membrane-bound protein by NMR spectroscopy.

ABSTRACT: Chemical shift anisotropy (CSA) tensors are essential in the structural and dynamic studies of proteins using NMR spectroscopy. Results from relaxation studies in biomolecular solution and solid-state NMR experiments on aligned samples are routinely interpreted using well-characterized CSA tensors determined from model compounds. Since CSA tensors, particularly the (15)N CSA, highly depend on a number of parameters including secondary structure, electrostatic interaction, and the amino acid sequence, there is a need for accurately determined CSA tensors from proteins. In this study, we report the backbone amide-(15)N CSA tensors for a 16.7-kDa membrane-bound and paramagnetic-heme containing protein, rabbit Cytochrome b(5) (cytb(5)), determined using the (15)N CSA/(15)N-(1)H dipolar transverse cross-correlation rates. The mean values of (15)N CSA determined for residues in helical, sheet, and turn regions are -187.9, -166.0, and -161.1 ppm, respectively, with an overall average value of -171.7 ppm. While the average CSA value determined from this study is in good agreement with previous solution NMR experiments on small globular proteins, the CSA value determined for residues in helical conformation is slightly larger, which may be attributed to the paramagnetic effect from Fe(III) of the heme unit in cytb(5). However, like in previous solution NMR studies, the CSA values reported in this study are larger than the values measured from solid-state NMR experiments. We believe that the CSA parameters reported in this study will be useful in determining the structure, dynamics, and orientation of proteins, including membrane proteins, using NMR spectroscopy.

SUBMITTER: Pandey MK

PROVIDER: S-EPMC3381076 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Determination of 15N chemical shift anisotropy from a membrane-bound protein by NMR spectroscopy.

Pandey Manoj Kumar MK Pandey Manoj Kumar MK Vivekanandan Subramanian S Ahuja Shivani S Pichumani Kumar K Im Sang-Choul SC Waskell Lucy L Ramamoorthy Ayyalusamy A

The journal of physical chemistry. B 20120604 24

Chemical shift anisotropy (CSA) tensors are essential in the structural and dynamic studies of proteins using NMR spectroscopy. Results from relaxation studies in biomolecular solution and solid-state NMR experiments on aligned samples are routinely interpreted using well-characterized CSA tensors determined from model compounds. Since CSA tensors, particularly the (15)N CSA, highly depend on a number of parameters including secondary structure, electrostatic interaction, and the amino acid sequ ...[more]

PMID: 22620865

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Project description:(13)C and (15)N chemical shift (CS) interaction is a sensitive probe of structure and dynamics in a wide variety of biological and inorganic systems, and in the recent years several magic angle spinning NMR approaches have emerged for residue-specific measurements of chemical shift anisotropy (CSA) tensors in uniformly and sparsely enriched proteins. All of the currently existing methods are applicable to slow and moderate magic angle spinning (MAS) regime, i.e., MAS frequencies below 20 kHz. With the advent of fast and ultrafast MAS probes capable of spinning frequencies of 40-100 kHz, and with the superior resolution and sensitivity attained at such high frequencies, development of CSA recoupling techniques working under such conditions is necessary. In this work, we present a family of R-symmetry based pulse sequences for recoupling of (13)C∕(15)N CSA interactions that work well in both natural abundance and isotopically enriched systems. We demonstrate that efficient recoupling of either first-rank (σ(1)) or second-rank (σ(2)) spatial components of CSA interaction is attained with appropriately chosen γ-encoded RN(n)(v) symmetry sequences. The advantage of these γ-encoded RN(n)(v)-symmetry based CSA (RNCSA) recoupling schemes is that they are suitable for CSA recoupling under a wide range of MAS frequencies, including fast MAS regime. Comprehensive analysis of the recoupling properties of these RN(n)(v) symmetry sequences reveals that the σ(1)-CSA recoupling symmetry sequences exhibit large scaling factors; however, the partial homonuclear dipolar Hamiltonian components are symmetry allowed, which makes this family of sequences suitable for CSA measurements in systems with weak homonuclear dipolar interactions. On the other hand, the γ-encoded symmetry sequences for σ(2)-CSA recoupling have smaller scaling factors but they efficiently suppress the homonuclear dipole-dipole interactions. Therefore, the latter family of sequences is applicable for measurements of CSA parameters in systems with strong homonuclear dipolar couplings, such as uniformly-(13)C labeled biological solids. We demonstrate RNCSA NMR experiments and numerical simulations establishing the utility of this approach to the measurements of (13)C and (15)N CSA parameters in model compounds, [(15)N]-N-acetyl-valine (NAV), [U-(13)C, (15)N]-alanine, [U-(13)C,(15)N]-histidine, and present the application of this approach to [U-(13)C∕(15)N]-Tyr labeled C-terminal domain of HIV-1 CA protein.

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Determination of 15N chemical shift anisotropy from a membrane-bound protein by NMR spectroscopy.

Publications

Determination of 15N chemical shift anisotropy from a membrane-bound protein by NMR spectroscopy.

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