Enhanced Resolution and Coherence Lifetimes in the Solid-State

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Enhanced Resolution and Coherence Lifetimes in the Solid-State NMR Spectroscopy of Perdeuterated Proteins under Ultrafast Magic-Angle Spinning € mit Akbey,‡ Sascha Lange,‡ Lyndon Emsley,† and J
ozef R. Lewandowski,† Jean-Nicolas Dumez,† U ,‡ Hartmut Oschkinat* †

Universit
e de Lyon (CNRS/ENS Lyon/UCB Lyon1), Centre de RMN a tres hauts champs, 5 rue de la Doua, 69100 Villeurbanne, France ‡ Leibniz-Institut f€ur Molekulare Pharmakologie (FMP), Robert-R€ossle-Strasse 10, 13125 Berlin, Germany

bS Supporting Information ABSTRACT: We investigate the combined effect of perdeuteration and fast magic-angle spinning on the resolution and sensitivity of proton-detected protein NMR spectra and on coherence lifetimes. With 60 kHz spinning of a microcrystalline R-spectrin SH3 sample at a field strength of 23 T, a regime is attained where there is no substantial difference in resolution between perdeuterated samples with 10 or 100% protons at the exchangeable sites.1H resolution is then limited by inhomogeneous contributions. Upon fast spinning, the most dramatic line narrowing effects are observed for residues in the loop or bend regions of the protein, probably due to the removal of destructive dynamics effects. This investigation paves the way for using samples with 100% protons at the exchangeable sites in structure determination protocols, since all backbone amide sites can now contribute to the signal. SECTION: Kinetics, Spectroscopy

N

MR spectroscopy is unique in its ability to provide access to both the structure and dynamics of biological systems at atomic resolution.1
4 In this context, it has been shown that magic-angle spinning (MAS) NMR offers special opportunities in investigations of heterogeneous preparations such as protofibrils or small polydisperse heat shock proteins, for membrane proteins in lipid environments, for studying molecular dynamics, or electronic properties of metal centers.4
11 However, unlocking its full potential still requires considerable developments. In particular, a robust, operator-independent scheme for structure determination including three- and higher-dimensional NMR techniques correlating all backbone and even side-chain signals is urgently required. Such developments in MAS NMR are thus far hampered by insufficient resolution and sensitivity. Of particular need are CBCANH- and CBCA(CO)NH-type12 correlations as a basis for a general assignment concept. In such experiments, as in NMR of biomolecules in general, 1H is the nucleus of choice for detection because of its high magnetogyric ratio. For samples at natural abundance, however, the strong homonuclear dipolar couplings that exist between protons in solids lead to severely broadened signals in 1H MAS NMR spectra. Various approaches have been proposed to mitigate this problem, which rely either on a spectroscopic route, where the 1H
1H dipolar couplings are averaged using homonuclear decoupling techniques and/or fast MAS,13
15 or on a synthetic route, where the 1H
1H network is weakened by “isotopic dilution”.16
18 Recrystallizing a sample of r 2011 American Chemical Society

a protein fully deuterated at nonexchangeable sites from a mixture of D2O and H2O makes it possible to strongly dilute and control the percentage of protons at exchangeable sites. Aiming at experiments with a spinning frequency of 24 kHz, crystallization from 30 to 40% H2O yields the deuteration level that represents the best compromise between resolution and sensitivity.17,19 The use of perdeuteration in combination with back-exchange has led to many applications. They include the extraction of structural constraints from correlation experiments,20
25 the measurement of site-specific dynamics parameters,26
30 and the use of scalar-coupling-based coherence transfer.31
33 In this communication, we investigate the combined effects of isotopic dilution and elevated MAS frequency on the resolution and sensitivity of 1H-detected spectra, and concomitantly analyze the evolution of bulk 1H and 15N coherence lifetimes. We show that excellent resolution and sensitivity can be obtained by combining perdeuteration of the protein followed by back exchange of all exchangeable protons and 60 kHz MAS. The spinning-frequency dependence of the line widths and coherence lifetimes suggest that a limit is reached in which the 1H resolution is primarily limited not by homogeneous dipolar broadening but rather by inhomogeneous contributions. Received: June 22, 2011 Accepted: August 11, 2011 Published: August 11, 2011 2205

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Figure 2. 15N-filtered 1H 1D CP (a
d) and 15N-filtered refocused INEPT (e
h) MAS spectra of 100% H2O [U
2H,13C,15N]SH3, recorded with 24 (d, h), 40 (c, g), 50 (b, f) and 60 kHz (a, e) MAS frequencies.

Figure 1. 1H-detected 2D 15N
1H correlation spectra recorded at ωr/ 2π = 60 kHz and ω0H/2π =1 GHz for R-spectrin SH3 at different protonation levels: (a) [U
13C,15N]SH3, (b) 100% H2O [U
2H,13C,15N]SH3, (c) 10% H2O [U
2H,13C,15N]SH3. Panel d depicts selected slices along the 1H dimension. The slices for [U
13C,15N]SH3, 100% H2O [U
2H,13C,15N]SH3, and 10% H2O [U
2H,13C,15N]SH3 are represented respectively by gray, black, and red strokes. The effective sample temperature was ∼34 °C for all the experiments as measured by the chemical shift of H2O with respect to DSS.1 The heteronuclear polarization transfer is CP-based in panels a and b and INEPT-based in panel c. During 15N t1 evolution, 15 kHz slpTPPM (a) and 5 kHz WALTZ-16 (b) were used, and a hard π pulse in the middle of the evolution was used in panel c.

Figure 1 shows 1H-detected 15N
1H two-dimensional (2D) correlation spectra of [U
13C,15N]- and [U
2H,13C,15N]-labeled

R-spectrin SH3 domain samples with various protonation levels at exchangeable sites, applying 60 kHz MAS. The 62-residue SH3 domain of chicken R-spectrin has been very well characterized by solid-state NMR.33,34 Spinning the sample at a frequency of 60 kHz already yields a fairly well-resolved spectrum on the fully protonated sample (Figure 1a) (average linewidth ∼ 253 Hz), yet the benefit of the combination with perdeuteration is clearly apparent from Figure 1b (100% H2O [U
2H,13C,15N]SH3, i.e., a sample recrystallized from 100% H2O/0% D2O solution) and 1c (10% H2O [U
2H,13C,15N]SH3, i.e., a sample recrystallized from 10% H2O/90% D2O solution). The 1H line widths are 24
70 Hz (0.2
0.7 ppm) and 15N line widths are 17
58 Hz (0.02
0.06 ppm) for 10% H2O [U
2H,13C,15N] SH3. Notably, excellent resolution is obtained in both dimensions for 100% H2O [U
2H,13C,15N]SH3, with line widths of 27
49 Hz (0.03 to 0.05 ppm) and 9
36 Hz (0.1 to 0.4 ppm) for proton and nitrogen signals, respectively. Expansions of selected regions of Figure 1 can be found in the Supporting Information. These results strongly suggest that at 60 kHz MAS, samples with 100% protons at the exchangeable sites yield the best balance of resolution and sensitivity, as further analyzed below. A comparison of the one-dimensional (1D) 15N-filtered 1H spectra in Figure 2, the 2D 15N
1H spectra in Figure 1b
c, 1D slices in Figure 1d, and the data in Table 1 indeed shows that no significant increase in resolution is obtained when the percentage of protons at exchangeable sites is decreased to 10% under the experimental conditions used (see also Figure S3 (Supporting Information) for the ratio of 1H line widths between 100% and 10% H2O samples in the INEPT-based 2D correlation spectra). The fact that 100% of the exchangeable sites can be protonated without large losses of resolution is expected to be highly beneficial for structural studies employing three-dimensional (3D) techniques. If transfer pathways are employed that involve only one or the same amide proton, for a level of 30% backexchange (which is the optimal for experiments performed at 24 kHz), only 30% of the molecules contribute to the signal. CACBNH-type experiments are such examples that would benefit from 100% protons at the exchangeable sites. In a more extreme case with transfers between two different amide protons, the average cross-peak probability for a long-range HN
HN correlation observed when using a 30% H2O sample is slightly less than 10% of the probability of the analogous cross peak observed for a 100% H2O sample. Obviously, factors other than 2206

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Figure 3. Distribution of 1H line widths for 100% H2O [U
2H, 13 15 C, N]SH3 as a function of the spinning frequency for all the observed resonances in the 1H-detected 2D 15N
1H correlation spectra. Red and black curves respectively depict bulk 15N and 1H T20 without decoupling as a function of spinning frequency. The infinity sign indicates that the residue is broadened beyond detection.

the dilution level contribute to the overall sensitivity. For example, when experiments using 1.3 mm and 3.2 mm rotors are compared, the relative volumes contribute by a factor of about 15 to the relative sensitivity. With the optimal level of 30% re-exchange for samples in a 3.2 mm rotor, considering a maximum spinning frequency of 24 kHz, this factor is reduced to 5. Although this speaks at first sight for experiments using 3.2 mm rotors, we are certain that other contributions, such as the coil filling factors or faster recycling due to low-power decoupling and shorter 1H T1, reduce this factor even further. This is illustrated by the comparison between the 2D 15N
1H correlation experiments on a 100% H2O sample at 60 kHz and on a 10% H2O sample at 24 kHz in a 1.3 mm rotor. We record signal-tonoise ratios (SNRs) per unit time and per unit mass that are on average 27 times higher in the 100% H2O sample at 60 kHz, which contrasts with the factor of 10 expected from the proton concentration only. It can be noted that a further reduction of the recycle delays could be obtained with the use of paramagnetic dopants.35,36 The dramatic effects of increasing the MAS frequency have been characterized for a variety of samples and experimental conditions.37
41 For perdeuterated biomolecules, a significant increase in 1H resolution is also expected as the interactions in dipolar-coupling networks become progressively better averaged out at higher spinning frequencies. Such behavior can indeed be observed in spectra of perdeuterated R-spectrin SH3 with 100% 1 H at exchangeable sites, in Figures 2
4. Typical values of the line widths, measured on 1H-detected 15N
1H correlation spectra, are given in Table 1, and it can be seen that 1H resolution is improved by a factor of up to 3.6 for resonances that are visible both at 24 and 60 kHz (Figure 4). At moderate spinning frequencies, some resonances such as D2, R21, T37, and N38 are simply either not detectable or so broad that it is very difficult to reliably extract their line widths. Even more interestingly, the line widths observed with 60 kHz MAS seem to approach a lower bound, as illustrated in Figure 3, which shows the evolution of the line widths for all the observed resonances as the spinning frequency is increased. While there is a large spread of values at 24 kHz, the range decreases when the spinning frequency is increased. Figure 4 shows the evolution of the 1H line width as a function of the spinning frequency for each backbone residue. To our

Figure 4. 1H line widths for 100% H2O [U
2H,13C,15N]SH3 as a function of residue and spinning frequency, and effective coupling (root sum square of all 1H
1H couplings) for the protons 2 lower SNR than analogous INEPT-based 1D spectra, only INEPT-based 2D spectra were recorded. f Bulk measurements. a

significantly better than low power sequences such as slpTPPM41 or low power XiX,46 e.g., in a 100% H2O sample the bulk backbone 15N T20 is ∼31 ms without decoupling, ∼43 ms under slpTPPM, and ∼78 ms under 5 kHz WALTZ-16. In the case of the 10% H2O sample, the bulk backbone 15N T20 under 5 kHz WALTZ-16 is ∼250 ms versus ∼147 ms without decoupling. The comparison of Figure 5a,b shows that, for experiments recorded at 60 kHz MAS, the ratios between the SNR available from INEPT-based experiments and the SNR available from the CP-based experiment are related to the effect of the spinning frequency on the line width of a given resonance. Resonances for which faster MAS leads to significant narrowing are also characterized by higher SNR in the CP-based experiment. On the other hand, resonances that do not narrow significantly at faster MAS are characterized by better SNR available from INEPTbased experiments. As their relative efficiencies vary from one chemical site to another, INEPT- and CP-based experiments are complementary in terms of the observed resonances. In the case of R-spectrin SH3, the more mobile sites such as E3 and T4 are present in INEPT-based but not in CP-based spectra. Also, other sites of the N-terminus that are usually not observed in dipolar-couplingbased experiments, such as G5 and D2, are visible in CP-based experiments at 60 kHz. Moreover, three sites (D48 and two unidentified) that have never been reported to be seen in 1H-detected experiments, are present in CP-based 1H
15N 2D correlation spectra on 100% H2O [U
2H,13C,15N]SH3 at 60 kHz MAS. As a final note in the context of protein dynamics studies, the analysis of coherence lifetimes reveals the fact that even at a high level of proton dilution and with ultrafast MAS, the decay of transverse coherences seems to be still largely governed by a coherent contribution. This can be appreciated by considering the average 15N coherence lifetime in 10% H2O [U
2H,13C,15N]SH3 with 60 kHz

MAS. The value of ∼150 ms obtained in the absence of 1H decoupling is in striking contrast with the value of ∼250 ms obtained with 5 kHz WALTZ-16 decoupling (which is found to yield the longest lifetimes at 500 MHz, 25 °C). The measurement of incoherent transverse relaxation times in solid proteins thus still remains elusive. In summary, the combination of perdeuteration and ultrafast MAS yields conditions that are optimal for sensitivity and resolution simultaneously, and significantly enhances coherence lifetimes. The very fast spinning frequencies achieved with the 1.3 mm rotors (up to ∼65 kHz) now allow to study deuterated proteins 100% back exchanged in H2O with similar resolution to 10 or 30% H2O samples. Furthermore, and of similar importance, protein signals that are not efficiently narrowed by intermediate MAS due to effects of chemical exchange or dynamics in conventional experimental schemes are now accessible for correlation experiments through MAS at 60 kHz on a 1 GHz NMR spectrometer. The approach explored here is thus expected to be highly beneficial for structural studies and site-specific measurements of dynamics.

’ ASSOCIATED CONTENT Materials and methods. Table bS 1 Supporting Information. 15 1 of H line widths in 2D N
H correlation spectra of 100% H2O [U
2H,13C,15N]SH3 at ω0H/2π = 1 GHz. Expansions from spectra in Figure 1. Figure illustrating ratio of 1H line widths between 100% H2O and 10% H2O [U
2H,13C,15N]SH3. Comparison of 1H-detected CP-based 2D 15N
1H correlation spectra on 100% H2O [U
2H,13C,15N]SH3 at different spinning frequencies. 1H-detected 2D 15N
1H correlation spectra for 100% and 10% H2O [U
2H,13C,15N]SH3 recorded at ωr/2π = 60 kHz and ω0H/2π = 500 GHz. Comparison of 1H

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1H correlation spectrum for [U
13C,15N]GB1 recorded at ωr/ 2π = 60 kHz and ω0H/2π = 1 GHz. Overlay of 1H-detected INEPT-based 2D 15N
1H correlation spectra on 100% H2O [U
2H,13C,15N]SH3 recorded at ωr/2π = 60 kHz and ω0H/2π = 1 GHz. SNR in 0.5 h experiment as a function of residue. This material is available free of charge via the Internet at http://pubs.acs.org.

’ ACKNOWLEDGMENT We thank Anna Diehl and Kristina Rehbein for providing the SH3 samples. This work was supported in part by the Agence Nationale de la Recherche (ANR PCV 2007 Protein Motion), DFG (SFB 740), and the Access to Research Infrastructures activity in the seventh Framework Program of the EC (BioNMR 261863). J.R.L. was partly supported by EU IRG (PIRG03-GA2008-231026). ’ REFERENCES (1) Cavanagh, J.; Fairbrother, W. J.; Palmer III, A. G.; Rance, M.; Skelton, N. J. Protein NMR Spectroscopy: Principles & Practice, 2nd ed.; Academic Press Inc.: San Diego, CA, 2006. (2) Henzler-Wildman, K.; Kern, D. Dynamic Personalities of Proteins. Nature 2007, 450, 964–972. (3) Castellani, F.; van Rossum, B.; Diehl, A.; Schubert, M.; Rehbein, K.; Oschkinat, H. Structure of a Protein Determined by Solid-State Magic-Angle-Spinning NMR Spectroscopy. Nature 2002, 420, 98–102. (4) McDermott, A. Structure and Dynamics of Membrane Proteins by Magic Angle Spinning Solid-State NMR. Annu. Rev. Biophys. 2009, 38, 385–403. (5) Tycko, R. Solid-State NMR Studies of Amyloid Fibril Structure. Annu. Rev. Phys. Chem. 2011, 62, 279–299. (6) Wasmer, C.; Lange, A.; Van Melckebeke, H.; Siemer, A. B.; Riek, R.; Meier, B. H. Amyloid Fibrils of the HET-s(218
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