On the performance of spin diffusion NMR techniques in oriented solids: prospects for resonance assignments and distance measurements from separated local field experiments

Abstract

NMR spin diffusion experiments have the potential to provide both resonance assignment and internuclear distances for protein structure determination in oriented solid-state NMR. In this paper, we compared the efficiencies of three spin diffusion experiments: proton-driven spin diffusion (PDSD), cross-relaxation-driven spin diffusion (CRDSD), and proton-mediated proton transfer (PMPT). As model systems for oriented proteins, we used single crystals of N-acetyl-L-(15)N-leucine (NAL) and N-acetyl-L-(15)N-valyl-L-(15)N-leucine (NAVL) to probe long and short distances, respectively. We demonstrate that, for short (15)N/(15)N distances such as those found in NAVL (3.3 Å), the PDSD mechanism gives the most intense cross-peaks, while, for longer distances (>6.5 Å), the CRDSD and PMPT experiments are more efficient. The PDSD was highly inefficient for transferring magnetization across distances greater than 6.5 Å (NAL crystal sample), due to small (15)N/(15)N dipolar couplings (<4.5 Hz). Interestingly, the mismatched Hartmann-Hahn condition present in the PMPT experiment gave more intense cross-peaks for lower (1)H and (15)N RF spinlock amplitudes (32 and 17 kHz, respectively) rather than higher values (55 and 50 kHz), suggesting a more complex magnetization transfer mechanism. Numerical simulations are in good agreement with the experimental findings, suggesting a combined PMPT and CRDSD effect. We conclude that, in order to assign SLF spectra and measure short- and long-range distances, the combined use of homonuclear correlation spectra, such as the ones surveyed in this work, are necessary.

FIGURE 1

Pulse sequences used to correlate ¹⁵N/¹⁵N: A) proton driven spin diffusion (PDSD), B) cross-relaxation driven spin diffusion (CRDSD), and C) proton-mediated proton transfer (PMPT). Asterisks indicate pulses that were adjusted by 90° to acquire phase-sensitive data in the indirect dimension (ω1).

FIGURE 2

Comparison of spin diffusion experiments on NAL. Note that the peak naming convention (peaks a, b, c, d) is from the most downfield (peak a) to the most upfield (peak d). For example, the cross-peak at ω₂ = 167 ppm and ω₁ = 133 ppm is referred to as ab. The PDSD experiment utilized a 3 sec mixing time, the CRDSD experiment used a ¹⁵N spinlock field of 21 kHz, and the PMPT experiment used a Z-filter of 3 sec and ¹H and ¹⁵N spinlock fields of 30 kHz and 21 kHz, respectively. The CRDSD and PMPT experiment used a mixing time of 10 msec. 1D sections are taken from the dotted line in the 2D spectra. All 2D spectra are shown at the same contour level, allowing for a direct comparison of peak intensities.

FIGURE 3

Comparison of spin diffusion experiments on NAVL. Note that the peak naming convention (peaks a, b, c, d) is from the most downfield (peak a) to the most upfield (peak d). For example, the cross-peak at ω₂ = 152 ppm and ω₁ = 99 ppm is referred to as ac. The PDSD experiment utilized a 3 sec mixing time, the CRDSD experiment used a ¹⁵N spinlock field of 21 kHz, and the PMPT experiment used a Z-filter of 3 sec and ¹H and ¹⁵N spinlock fields of 65 kHz and 55 kHz, respectively. The CRDSD and PMPT experiment used a mixing time of 10 msec. 1D sections are taken from the dotted line in the 2D spectra. All 2D spectra are shown at the same contour level, allowing for a direct comparison of peak intensities.

FIGURE 4

PDSD experiments on the NAL single crystal. All 12 cross-peaks are shown from the spectra are shown in arbitrary intensity units. All experiments from the NAL crystal (Figures 6, 8, 10 and 11) are shown in the same relative units (i.e., same noise floor).

FIGURE 5

PDSD experiments on the NAVL single crystal. All 12 cross-peaks are shown from the spectra are shown in arbitrary intensity units. All experiments from the NAVL crystal (Figures 7, 9 and 12) are shown in the same relative units (i.e., same noise floor).

FIGURE 6

CRDSD experiments on the NAL single crystal. The ¹⁵N spinlock field was varied while keeping the mixing time fixed at 10 msec. The intensities are expressed in arbitrary units. All experiments were acquired in an interleaved manner to avoid potential differences in the experiments, and are therefore relevant to compare within each figure.

FIGURE 7

CRDSD experiments on the NAVL single crystal. The ¹⁵N spinlock field was varied while keeping the mixing time fixed at 10 msec. The intensities are expressed in arbitrary units.

FIGURE 8

CRDSD experiments on the NAL single crystal. The ¹⁵N spinlock field was fixed at 21 kHz, while the mixing time was varied between 1–20 msec. The cross-peak intensities are plotted in arbitrary units.

FIGURE 9

CRDSD experiments on the NAVL single crystal. The ¹⁵N spinlock field was fixed at 21 kHz, while the mixing time was varied between 1–20 msec. The cross-peak intensities are plotted in arbitrary units.

FIGURE 10

PMPT experiments on the NAL single crystal. The ¹H spinlock field was varied between 0–70 kHz for three different ¹⁵N spinlock fields (21, 37.5, and 52.5 kHz). The values are also plotted as the Hartmann-Hahn mismatch percentage. The mixing time was fixed at 10 msec for all points. Cross-peak intensities are plotted in arbitrary units. The dip in the curves is due to the Hartmann-Hahn match. The other cross-peaks are essentially the same as that plotted for the cross-peak ad. All other curves are shown in Supplementary Figures 17 and 18.

FIGURE 11

PMPT experiments on the NAL single crystal. The ¹H spinlock field was set to 32.5, 47.5 and 62.5 kHz for 21, 37.5 and 52.5 kHz ¹⁵N spinlock, respectively, based on the results shown in Figure 10. The mismatched Hartmann-Hahn (MMHH) mixing time was varied from 1–20 msec. The cross-peak intensities are in arbitrary units (i.e., not divided by the diagonal peak intensity).

FIGURE 12

PMPT experiments on the NAVL single crystal. The ¹H spinlock field was varied for a ¹⁵N spinlock of 57.5 kHz. The mixing time was fixed at 10 msec for all points. The curves plotted are for crosspeaks ac (intramolecular ¹⁵N/¹⁵N, 3.3 Å) and ad (intermolecular ¹⁵N/¹⁵N, > 6 Å). Intensities are plotted in arbitrary units. The dip in the curves is due to the Hartmann-Hahn match. The dotted line indicates the best transfer ¹H spinlock value for intermolecular transfer in NAVL. The optimal value for intermolecular transfer results in less magnetization transfer for intramolecular sites. The other cross-peaks are essentially the same as those plotted for the cross-peaks ac and ad. All other curves are shown in Supplementary Figures 18 and 19.

FIGURE 13

Twelve-spin simulations of the PMPT and CRDSD experiments on the NAL single crystal. A) Varying the spinlock field on the ¹H spinlock field for three different ¹⁵N spinlock fields (17.5, 33, and 50 kHz) at a MMHH mixing time of 20 msec. Note that for a ¹H spinlock field of 0 kHz, this is the CRDSD experiment for a 17.5 kHz ¹⁵N spin-lock and the RFDSD experiment for larger ¹⁵N spin-locks. B) Build-up for the most optimal transfer efficiencies in panel A. For ¹⁵N spinlocks of 17.5, 33, and 50 kHz, the ¹H spinlock field was maximal at 22.5, 40, and 55 kHz, respectively. C) Several mixing time build-up curves for different ¹H spinlock fields when the ¹⁵N spinlock was set to 17.5 kHz.

Figure 14

Comparison of PDSD, CRDSD, and PMPT experiments for NAL and NAVL normalized for the mixing time in the PDSD experiment. Since a 3 sec recycle delay was used for every experiment, normalization was done by dividing the PDSD 2D peak intensities by 1.29, 1.53, 2.08, and 3.32 to account for 2, 4, 10, and 30 sec mixing times, respectively. Although the PMPT experiment used a 3 sec Z-filter time, no normalization was done for this experiment due to the fact that a much shorter time can be used that would only marginally influence the experimental acquisition time. For NAVL, much of the magnetization created in the PMPT experiment originated from the 3 sec Z-filter time. Circles represent all cross peaks observed in the NAL spectra and only intramolecular ¹⁵N/¹⁵N cross-peaks in the NAVL crystal (i.e., peaks ac, ca, bd, and db). Triangles represent the average of the cross-peak intensities shown (values in Table I). The CRDSD experimental cross-peaks are from the spectra using a 10 msec mixing time with a ¹⁵N spinlock of 21 kHz (both NAL and NAVL). The PMPT experimental cross-peaks are from the 10 msec mixing time with the indicated ¹⁵N spinlock for NAL and the optimized ¹H spinlock used in Figure 11. For NAVL, the PMPT experiment used the 10 msec mixing time with ¹H and ¹⁵N spinlocks of 65.8 and 57.5 kHz, respectively (same as in Supplementary Figure 19). It is not appropriate to compare the intensities between NAL and NAVL, as the crystal sizes were different.