High-resolution solid-state NMR structure of alanyl-prolyl-glycine

Abstract

We present a de novo high-resolution structure of the peptide Alanyl-Prolyl-Glycine using a combination of sensitive solid-state NMR techniques that each yield precise structural constraints. High-quality (13)C-(13)C distance constraints are extracted by fitting rotational resonance width (R(2)W) experiments using Multimode Multipole Floquet Theory and experimental chemical shift anisotropy (CSA) orientations. In this strategy, a structure is first calculated using DANTE-REDOR and torsion angle measurements and the resulting relative CSA orientations are used as an input parameter in the (13)C-(13)C distance calculations. Finally, a refined structure is calculated using all the constraints. We investigate the effect of different structural constraints on structure quality, as determined by comparison to the crystal structure and also self-consistency of the calculated structures. Inclusion of all or subsets of these constraints into CNS calculations resulted in high-quality structures (0.02A backbone RMSD using all 11 constraints).

Fig. 1

Crystal structure of APG [14] showing the 11 SSNMR experimental constraints used in the structural refinement. Torsion angles are displayed in violet, REDOR distances in orange and R²W constraints in black. Note that the proline ring has a split occupancy between two conformers in the crystal structure. (For interpretation of colour mentioned in this figure, the reader is referred to the web version of this article.)

Fig. 2

(a) Relative orientations of the Proline C′ and C^γ CSAs are shown superimposed on the initial structure calculated using REDOR and torsion angle constraints. (b) Histogram displaying the range of Pro C′-Pro C^γ distances calculated from an ensemble of accessible structures generated using simulated annealing (CNS) without any constraints. X-ray (x) and reported NMR (o) distances are indicated. This graphic was produced with SIMMOL [17].

Fig. 3

Crystal lattice of APG showing competing intermolecular distances to the long-range internuclear distance that could be used for structural refinement.

Fig. 4

Twenty lowest energy structures of APG using simulated annealing (CNS) (a) no constraints (b) 2 ψ angles from NCCN measurements (c) 4 ¹³C–¹³C distance constraints (d) 4 ¹³C–¹⁵N heteronuclear constraints (e) all 11 constraints (f) crystal structure.

Fig. 5

(a) Contour plot of the root mean square deviation between experimental and simulated R²W profiles for P_O to P_γ as a function of distance and relaxation. (b) Magnetization transfer from P_o to P_γ in R²W as a function of spinning frequency. The experimental data is shown in blue, and the fit in green. (For interpretation of colour mentioned in this figure, the reader is referred to the web version of this article.)

Fig. 6

The χ² surface for azimuthal (α) and polar (β) angles of the CSA-dipole orientation with each contour level two times the previous level. Labeled APG was diluted to 10% in natural abundance APG and data were collected at 14.1 Tesla with an MAS spinning frequency of 8929 Hz. The superimposed dashed line shows the allowed azimuthal and polar angles corresponding to a rotation about the terminal ψ dihedral angle, with the best-fit at α = 31°, β = 90°.

Fig. 7

R²W spectra at five spinning frequencies. The P_γ crosspeak intensifies as the spinning frequency is swept the R² condition between the spin pair.

Fig. 8

Dephasing curves for extraction of Ψ torsion angle. An N_GlyCCN_Pro Ψ angle of 152° (blue) was extracted compared to 153° from the X-ray data and N_AlaCCN_Gly Y angle was found to be 162° (red) versus 157° from the X-ray data. SPINEVOLUTION [27] was used to fit the data. (For interpretation of colour mentioned in this figure, the reader is referred to the web version of this article.)