Journal
JOURNAL OF MAGNETIC RESONANCE
Volume 259, Issue -, Pages 225-231Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.jmr.2015.08.019
Keywords
Static NMR; N-15 CSA; SAMMY; Protein dynamics
Funding
- National Science Foundation [MCB-1122154, DMR-1157490]
- National Institutes of Health [P20GM103395]
- State of Florida
- U.S. Department of Energy
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In this work, we assess the usefulness of static N-15 NMR techniques for the determination of the N-15 chemical shift anisotropy (CSA) tensor parameters and N-15-H-1 dipolar splittings in powder protein samples. By using five single labeled samples of the villin headpiece subdomain protein in a hydrated lyophilized powder state, we determine the backbone N-15 CSA tensors at two temperatures, 22 and 35 degrees C, in order to get a snapshot of the variability across the residues and as a function of temperature. All sites probed belonged to the hydrophobic core and most of them were part of alpha-helical regions. The values of the anisotropy (which include the effect of the dynamics) varied between 130 and 156 ppm at 22 degrees C, while the values of the asymmetry were in the 0.32-0.082 range. The Leu-75 and Leu-61 backbone sites exhibited high mobility based on the values of their temperature-dependent anisotropy parameters. Under the assumption that most differences stem from dynamics, we obtained the values of the motional order parameters for the N-15 backbone sites. While a simple one-dimensional line shape experiment was used for the determination of the N-15 CSA parameters, a more advanced approach based on the magic sandwich SAMMY pulse sequence (Nevzorov and Opella, 2003) was employed for the determination of the N-15-H-1 dipolar patterns, which yielded estimates of the dipolar couplings. Accordingly, the motional order parameters for the dipolar interaction were obtained. It was found that the order parameters from the CSA and dipolar measurements are highly correlated, validating that the variability between the residues is governed by the differences in dynamics. The values of the parameters obtained in this work can serve as reference values for developing more advanced magic-angle spinning recoupling techniques for multiple labeled samples. (C) 2015 Elsevier Inc. All rights reserved.
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