| Autor: |
Nieuwkoop, Andrew J., Wylie, Benjamin J., Franks, W. Trent, Shah, Gautam J., Rienstra, Chad M. |
| Předmět: |
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| Zdroj: |
Journal of Chemical Physics; 9/7/2009, Vol. 131 Issue 9, p095101, 8p, 1 Diagram, 2 Charts, 7 Graphs |
| Abstrakt: |
We show that quantitative internuclear 15N–13C distances can be obtained in sufficient quantity to determine a complete, high-resolution structure of a moderately sized protein by magic-angle spinning solid-state NMR spectroscopy. The three-dimensional ZF-TEDOR pulse sequence is employed in combination with sparse labeling of 13C sites in the β1 domain of the immunoglobulin binding protein G (GB1), as obtained by bacterial expression with 1,3-13C or 2-13C-glycerol as the 13C source. Quantitative dipolar trajectories are extracted from two-dimensional 15N–13C planes, in which ∼750 cross peaks are resolved. The experimental data are fit to exact theoretical trajectories for spin clusters (consisting of one 13C and several 15N each), yielding quantitative precision as good as 0.1 Å for ∼350 sites, better than 0.3 Å for another 150, and ∼1.0 Å for 150 distances in the range of 5–8 Å. Along with isotropic chemical shift-based (TALOS) dihedral angle restraints, the distance restraints are incorporated into simulated annealing calculations to yield a highly precise structure (backbone RMSD of 0.25±0.09 Å), which also demonstrates excellent agreement with the most closely related crystal structure of GB1 (2QMT, bbRMSD 0.79±0.03 Å). Moreover, side chain heavy atoms are well restrained (0.76±0.06 Å total heavy atom RMSD). These results demonstrate for the first time that quantitative internuclear distances can be measured throughout an entire solid protein to yield an atomic-resolution structure. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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