An experimental strategy has been developed to increase the efficiency of dynamic nuclear polarization (DNP) in solid-state NMR studies. The method makes assignments simpler, faster, and more reliable via sequential correlations of both side-chain and Cα resonances. The approach is particularly suited to complex biomolecules and systems with significant chemical-shift degeneracy. It was designed to overcome the spectral congestion and line broadening that occur due to sample freezing at the cryogenic temperatures required for DNP. Nonuniform sampling (NUS) is incorporated to achieve time-efficient collection of multidimensional data. Additionally, fast (25 kHz) magic-angle spinning (MAS) provides optimal sensitivity and resolution. Data collected in <1 wk produced a virtually complete de novo assignment of the coat protein of Pf1 virus. The peak positions and linewidths for samples near 100 K are perturbed relative to those near 273 K. These temperature-induced perturbations are strongly correlated with hydration surfaces.
DNP; Pf1 bacteriophage; SSNMR.
Conflict of interest statement
The authors declare no conflict of interest.
A) The 2D 13C– 13C DARR spectra (100 ms mixing time) of Pf1 illustrating linewidth differences between DNP conditions at 106 K (purple) and the same sample at 273 K (green). ( B) Expansions around several assigned peaks with unique chemical shifts are shown. Although some well-resolved peaks at 273 K remained well resolved at 106 K, peaks in more crowded regions can become unrecognizable. Even for well-resolved peaks, linewidths broadened approximately twofold, from <100 Hz (0.5 ppm) to >200 Hz (1.3 ppm), making assignments more challenging. See SI Appendix for additional experimental details, including demonstration of 35-fold enhancement in 3.2-mm rotor and 60-fold enhancement in 1.9-mm rotor ( SI Appendix, Fig. S1).
A) Contrast of the polarization transfer pathways ( Upper) and key chemical-shift correlations ( Lower) for NCACX/NCOCA and S 3 assignment strategies, as depicted on a protein backbone. Regions of overlap between the forward and backward correlation experiments for each assignment strategy are shown in dashed outlines. The S 3 approach covers the full transfer pathway of both NCACX and NCOCA experiments in a single experiment and provides a much larger overlap region, enabling multiple chemical-shift correlations between neighboring residues (e.g., N–Cα–N and direct Cα–Cα) for maximum robustness and ease of assignment. ( B and C) Schematics of the pulse sequences for backward [ B; 4D CX(Cα)NC′CX i→ i − 1] and forward [ C; 3D CX(C′)N(Cα)CX i→ i + 1] S 3 correlation experiments. In both experiments, a short (60–80 us) H–C tangential CP was used to maximize side-chain 13C polarization, before a short 13C– 3C homonuclear recoupling period to maximize transfer to Cα or C′. Ten milliseconds of DARR mixing was typically used for Cα, and 20 ms for C′ (empirically optimized). To transfer polarization to the previous or subsequent residue, 13C– 15N SPECIFIC-CP (Cα–N in the backward case, and C′ –N in the forward case), a 15N chemical shift encoding period, followed by 15N– 13C SPECIFIC-CP (N–C′ in the backward case, N–Cα in the forward case) was used. After optional 13C chemical-shift encoding on Cα/C′, a second 13C– 13C homonuclear recoupling element transferred polarization to the side chain, which was detected directly. This transfer required longer mixing times to achieve several-bond transfers; 50 ms of DARR mixing was used for short-range correlations and 250 ms for long-range correlations. Spinal-64 decoupling (dec) was used during evolution periods; continuous wave (CW) decoupling was applied during cross-polarization.
3 spectra of Pf1 enabled assignment of distant side-chain sites, whereas comparison with short-range S 3 helped to identify nearest-neighbor backbone correlations, as shown in 13C– 13C planes ( A) and 13C– 15N planes ( B) at the indicated 15N and 13C indirect dimension frequency, respectively, from short-range (green) and long-range (orange) CX(Cα)N(C′)CX i→ i − 1 S 3 spectra. DARR transfers of 10 and 50 ms were used for the short-range experiment; 50- and 250-ms DARR were used for long-range S 3. 13C and 15N linewidths were as low as 1.0 and 3.5 ppm, respectively. See SI Appendix for additional experimental details.
Strip plot of assignments for residues 21–29 of the Pf1 coat protein, showing representative backbone and side-chain interresidue “walks.” Data from 50-ms NCACX (blue), “short-range” S
i→ i − 1 10,50 ms (green), and “long-range” S 3
i→ i − 1 50, 250 ms (orange) are overlaid at the indicated 15N frequency in each slice. Other spectra, including NCOCA and S 3
i→ i + 1, were used to confirm and extend these assignments. See SI Appendix for more experimental details.
Sequential assignment of A36–L38 shown in
13C– 13C slices of 3D NCACX (green), 3D S 3
i→ i + 1 (purple), and i→ i − 1 (orange) at the indicated 15N chemical shifts. Notably, the two S 3 spectra show very different patterns. ( A) In S 3
i→ i − 1, G37-A36 cross-peaks appeared at the glycine frequency in the indirect dimension ( y axis) and were “read out” at alanine frequencies in the direct dimension ( x axis), whereas in S 3
i→ i + 1, these frequencies were reversed. No S 3
i→ i − 1 intensity was detected at the glycine frequency, and no S 3
i→ i + 1 intensity was observed on the alanine slice, except for a diagonal peak representing G37Cα–N–Cα. ( B) A similar pattern was observed for G37–L38 interresidue cross-peaks. ( C) With the exception of some diagonal intensity, the observed cross-peak patterns were exactly as expected from the transfer diagram, demonstrating the excellent selectivity of S 3 experiments. See SI Appendix for additional experimental details.
A) Mappings of average 13C and 15N CSP for each residue of the Pf1 coat protein. Signed CSPs and absolute value CSPs (|CSP|) were averaged over all atoms of each residue; CSPs were computed as σ 106
K – σ 273
K in all cases. The shade and intensity of color correspond to perturbation levels, as indicated. A plot of residue average CSPs by residue number is shown in SI Appendix, Fig. S7. Large CSPs are clustered in three main regions: the outward-facing N terminus, a central kink region, and the inward-facing C terminus, which is the DNA interaction domain. ( B) An independent map of where water molecules contact the protein at 278 K shows very similar patterns (52) on a single copy of the coat protein as well as on sections of the Pf1 assembly looking perpendicular to and down the central symmetry axis. The high degree of correlation suggests that solvent exposure represents a significant driving force of chemical shift changes in moving from conditions near 273 K to DNP conditions near 100 K.
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Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.
Bacteriophage Pf1 / chemistry*
Bacteriophage Pf1 / metabolism
Nuclear Magnetic Resonance, Biomolecular*
Pseudomonas aeruginosa / virology*