Nanomole-scale protein solid-state NMR by breaking intrinsic 1HT1 boundaries

Abstract

We present an approach that accelerates protein solid-state NMR 5-20-fold using paramagnetic doping to condense data-collection time (to approximately 0.2 s per scan), overcoming a long-standing limitation on slow recycling owing to intrinsic (1)H T(1) longitudinal spin relaxation. Using low-power schemes under magic-angle spinning at 40 kHz, we obtained two-dimensional (13)C-(13)C and (13)C-(15)N solid-state NMR spectra for several to tens of nanomoles of beta-amyloid fibrils and ubiquitin in 1-2 d.

Figure 1

The effectiveness of the PACC approach for hydrated proteins. (a) Comparison of superimposed 2D ¹³C/¹³C chemical-shift correlation SSNMR spectra of uniformly ¹³C-labeled ubiquitin (1.8 mg) in microcrystals in the (black) absence and (red) presence of 10 mM Cu(II)-EDTA respectively obtained in standard and PACC approaches at a ¹H frequency of 400.2 MHz, together with (b) slices at selected positions. (c) A proposed mechanism of paramagnetic ¹H T₁ relaxation enhancement for hydrated protein microcrystals by Cu(II)-EDTA doping. (d) Comparison of 2D ¹³C/¹³C SSNMR spectra of fibrillized ¹³C- and ¹⁵N-labeled 40-residue Alzheimer’s β-amyloid (Aβ(1–40); 1.4 mg) that were obtained in the (red) presence and (black) absence of 200 mM Cu-EDTA. All the experiments in Fig. 1 were performed at a spinning speed of 40 kHz with signal acquisitions under low-power TPPM decoupling at rf-fields of 7 kHz. The mixing time for the fpRFDR sequence was 1.6 ms. See Supplementary Methods and Supplementary Figs. 1, 2, 6 online for sample preparations, signal assignments for ubiquitin, pulse sequences, and other details.

Figure 2

2D chemical-shift correlation SSNMR spectra for mass-limited systems by the PACC approach. (a) A 2D ¹³C/¹³C correlation SSNMR spectrum obtained by PACC for fibrillized uniformly ¹³C- and ¹⁵N-labeled 42-residue Alzheimer’s β-amyloid (Aβ) peptide (0.4 mg or 80 nmol) doped with 200 mM Cu-EDTA, together with preliminary assignments based on amino-acid types for ¹³C_α/¹³C_β cross peaks (dotted circles) and ¹³C_α/¹³CO (dotted squares). The recycle delays were set to 180 ms, which is approximately three times the ¹H T₁ value (~ 60 ms). The presence of strong cross peaks for Ala-42 (orange arrows) suggest the ordered structure at the C-terminal of Aβ(1–42). (b) Nano-mole scale analysis by 2D ¹³CO/¹⁵N correlation SSNMR of uniformly ¹³C- and ¹⁵N-labeled ubiquitin (22 nmol or 200 μg) in microcrystals doped with 10 mM Cu-EDTA. The experimental time was only 2.7 h with recycle delays of 165 ms (¹H T₁ ~ 55 ms). After the first cross-polarization, ¹⁵N signals were observed during the t₁ period (t₁^max = 12 ms) under low-power TPPM decoupling sequence. The real or imaginary component of the signal was transferred to ¹³CO by double-quantum ¹³C-¹⁵N ramped cross polarization, in which the rf-intensity for ¹³C (ω_C/2π) was swept from 27 to 23 kHz while that for ¹⁵N (ω_N/2π) was fixed at 15 kHz during the contact time of 5 ms. See the Supplementary Methods and Supplementary Fig. 1 online for further details about the pulse sequences and sample preparation.

Figure 3

¹³C T₁ relaxation rate enhancement (ΔR₁) by 200 mM Cu-EDTA paramagnetic relaxation agent on Aβ(1–40) fibrils. The data were collected for three Aβ samples labeled with uniformly ¹³C- and ¹⁵N-labeled amino acids at selected sites in different schemes as (1) Ala-2, Phe-4, Gly-9, Val-18, (2) Val-18, Phe-20, Ala-21, Ile-31, Gly-33 and (3) Ala-30, Ile-32, Gly-38, Val-39. ¹³C longitudinal relaxation rates R₁ were measured in the presence (R₁′) and absence (R₁^dia) of Cu-EDTA by inversion recovery experiments detected in 1D ¹³C CPMAS spectra at a spinning speed of 40 kHz at ¹H frequency of 400.2 MHz. ΔR₁ was obtained from ΔR₁ = R₁′ − R₁^dia. The color coding corresponds to the data for the unstructured N-terminal region (yellow: Residue 1–9), the N-terminal β-sheet region (red: Residue 10–22), and the C-terminal β-sheet region (blue: Residue 30–40). The structural model based on ref. in the inset is also color coded as described above with the loop region (Residue 23–29), which is also denoted in yellow. See the Supplementary Methods online for further details about the experiments and the sample preparation.