Protons as Versatile Reporters in Solid-State NMR Spectroscopy

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Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX

Protons as Versatile Reporters in Solid-State NMR Spectroscopy Suresh K. Vasa,† Petra Rovó,† and Rasmus Linser* Department Chemistry, Ludwig-Maximilians-University Munich, Butenandtstr. 5-13, 81377 Munich, Germany Center for Integrated Protein Science, Ludwig-Maximilians-Universität, 81377 Munich, Germany CONSPECTUS: Solid-state nuclear magnetic resonance (ssNMR) is a spectroscopic technique that is used for characterization of molecular properties in the solid phase at atomic resolution. In particular, using the approach of magic-angle spinning (MAS), ssNMR has seen widespread applications for topics ranging from material sciences to catalysis, metabolomics, and structural biology, where both isotropic and anisotropic parameters can be exploited for a detailed assessment of molecular properties. High-resolution detection of protons long represented the holy grail of the field. With its high natural abundance and high gyromagnetic ratio, 1H has naturally been the most important nucleus type for the solution counterpart of NMR spectroscopy. In the solid state, similar benefits are obtained over detection of heteronuclei, however, a rocky road led to its success as their high gyromagnetic ratio has also been associated with various detrimental effects. Two exciting approaches have been developed in recent years that enable proton detection: After partial deuteration of the sample to reduce the proton spin density, the exploitation of protons could begin. Also, faster MAS, nowadays using tiny rotors with frequencies up to 130 kHz, has relieved the need for expensive deuteration. Apart from the sheer gain in sensitivity from choosing protons as the detection nucleus, the proton chemical shift and several other useful aspects of protons have revolutionized the field. In this Account, we are describing the fundamentals of proton detection as well as the arising possibilities for characterization of biomolecules as associated with the developments in our own lab. In particular, we focus on facilitated chemical-shift assignment, structure calculation based on protons, and on assessment of dynamics in solid proteins. For example, the proton chemical-shift dimension adds additional information for resonance assignments in the protein backbone and side chains. Chemical shifts and high gyromagnetic ratio of protons enable direct readout of spatial information over large distances. Dynamics in the protein backbone or side chains can be characterized efficiently using protons as reporters. For all of this, the sample amounts necessary for a given signal-to-noise have drastically shrunk, and new methodology enables assessment of molecules with increasing monomer molecular weight and complexity. Taken together, protons are able to overcome previous limitations, by speeding up processes, enhancing accuracies, and increasing the accessible ranges of ssNMR spectroscopy, as we shall discuss in detail in the following. In particular, these methodological developments have been pushing solid-state NMR into a new regime of biological topics as they realistically allow access to complex cellular molecules, elucidating their functions and interactions in a multitude of ways.

1. INTRODUCTION

volume would have prevented use of the amounts of material that were commonly used for reasonable signal-to-noise. Successful proton detection was eventually achieved by continuous efforts of multiple laboratories in the early 2000s, in particular, the Reif and Zilm laboratories by diluting the proton concentration in the protein by extensive deuteration1−3 as well as the Rienstra, Tycko, and Ishii laboratories, putting faster MAS forward.4−6 Several additional laboratories (Meier, Pintacuda, Lewandowski, and others) have pushed proton detection further since.7−10 At slow MAS frequencies (∼14 kHz), a protonation of ∼10% of the exchangeable protons in an otherwise completely deuterated protein results in ca. 20 Hz proton line width.2,11,12 Similarly, 24 kHz MAS and ∼30%

Solid-state nuclear magnetic resonance spectroscopy has long relied on comparably insensitive heteronuclear (for example, 13 C and 15N) detection, whereas solution NMR has always made extensive use of protons, yielding maximal sensitivity due to their natural abundance and high gyromagnetic ratio. The associated strong magnetic dipole moment, however, also entails strong homonuclear proton−proton dipolar interactions, which leads to homogeneously broadened lines if not averaged out sufficiently by molecular reorientation or magicangle spinning (MAS). Rotor diameters used for the common, 13 C-detected techniques did not allow spinning fast enough to average out these interactions. Smaller rotors (≤1.3 mm outer diameter), which might have enabled sufficiently fast spinning, on the other hand, did not seem very practical: Their reduced © XXXX American Chemical Society

Received: February 1, 2018

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DOI: 10.1021/acs.accounts.8b00055 Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Accounts of Chemical Research proton back-exchange results in akin narrow line widths.13−15 The development of faster rotors has since allowed the decrease of proton dilution at maintained homogeneous proton line widths. At 60 kHz MAS, samples are commonly deuterated and 100% back-exchanged7,16 at amide sites, or they are protonated only at methyl sites (using CHD2 labeling)8 or other aliphatic sites via random adjoined protonation (RAP)17 or fractional deuteration.18 Without deuteration, homogeneous line widths of ∼250 Hz (60 kHz)19,20 or ∼80 Hz (110 kHz MAS)9,21 can be achieved, which may or may not be sufficient given the broad natural (inhomogeneous) line widths of noncrystalline preparations.22,23 The apparent disadvantage of small maximal sample volumes in fast-MAS rotors has been counteracted by two factors, namely, the influence of the gyromagnetic ratio γ (γ3/2 proportionality of the detected signal, assuming no other differences)24 and by the increased coil efficiency (K = B1e/√P, with B1e being the induced magnetic field and P the applied rf power) of smaller coils, where K is proportional to the square root of Q, the quality factor of the respective rf channel, and inversely proportional to the square root of VS, the sample volume. One may define a so-called sensitivity index KVS as a simplified measure proportional to the signal-to-noise ratio. As a result, sensitivity becomes roughly proportional to the coil diameter, rather than the volume.25 Even though practically, additional effects play a role, like the homogeneous and inhomogeneous line widths of the signal and how well a probe is actually built, the ca. 7-fold lower sensitivity index of a 1.3 mm coil (compared to 4 mm for Bruker systems) is fully compensated by switching to proton detection.16,26 For fully protonated proteins in faster spinning/smaller rotors, although the achievable signal-to-noise slightly decreases, the potential dispensability of deuteration as well as the additional technical prospects compensate for this drawback.21 Figure 1 visualizes

milliseconds (Figure 2E and D) after paramagnetic doping.11,27 Also the duration of blocks of transverse magnetization can be drastically increased, facilitating scalar transfers between various nuclei.12,15,28,29 In a 15N−13C INEPT transfer for example, more than 30 ms of transverse magnetization are usually necessary. Even protons themselves can be involved in INEPT transfers like in solution NMR.12 This enables detection of residues of various degrees of mobility at the same time, whereas in classical approaches only rigid (accessible via dipolar transfers) or highly mobile residues (via INEPTs, without proton decoupling) could be observed.29 Figure 2A and B shows a comparison of dipolar and scalar transfers in an H/N correlation of the microcrystalline SH3 domain of chicken αspectrin. Under the conditions of a reduced proton dipolarcoupling network, essentially all solution pulse sequence elements are accessible for solid-state NMR. This includes TROSY coherence selection, useful for characterization of dynamics via differential relaxation and for assignment of residues moving on the intermediate-time scale regime.29,30 In addition, water interactions like solvent magnetization transfer and H/D exchange can be probed directly and with high spatial and time resolution.31 Figure 2C shows an SH3 sample exchanged with D2O over 1 d compared to a reference spectrum. Technically, in conventional approaches, strong proton dipolar couplings can be exploited to connect carbons via proton-driven spin diffusion32 and its variants. In their absence, direct homonuclear recoupling like RFDR33 and HORROR34 or scalar mixing methods like TOBSY35 and MOCCA36 can be used as alternatives. The latter approaches enable (throughbond) correlations along an entire side chain in the absence of spin diffusion. Proton detection also requires water suppression, which sets some constraints for the architecture of pulse sequences. Water suppression is usually achieved using approaches like MISSISSIPPI,37 which saturates water while storing magnetization of interest in a heteronucleus. We will elaborate in the following how these features affect the design of pulse schemes for assignment, structure, and dynamics, and can be used even for complex molecules like the 29 kDa enzyme human carbonic anhydrase II (hCAII; see an assigned H/N correlation in Figure 2F).26

3. ASSIGNMENT EXPERIMENTS For conventional 13C-detected approaches, chemical shifts give direct information about the residue type and backbone dihedral angles, and interresidual correlations are within easy reach. As such, 2D spectra have widely been employed for resonance assignment there. By contrast, neither the amide proton nor the adjacent 15N yield information for sequential backbone assignment. Proton-detected assignment experiments are hence usually recorded using three or more dimensions in order to incorporate backbone carbons. Established 1H-detected 3D assignment experiments are usually similar to solution NMR methods, transferring magnetization from protons to carbons and back via 15N in an out-and-back fashion.12,15,28,38 Examples include the hCONH, hCANH, hCAcoNH, hCOcaNH, hcaCBcaNH, and hcaCBcacoNH experiments. These sequences are modified according to whether aliphatic protons are present for initial magnetization transfer or not and can be recorded with dipolar transfers throughout, using INEPTs only for CC transfers, or for both CC and NC transfers, or INEPT transfers exclusively. (See an elaborate comparison of efficiencies for a deuterated

Figure 1. Different types of protons in proteins and their use for various purposes. Whereas amide protons have been used for sequential chemical-shift assignments, structure calculation, and backbone dynamics, methyl groups have played a role predominantly for structure calculation and side chain dynamics. Hα protons have recently been suggested for facilitating backbone assignments.21

some spectroscopic possibilities arising from different kinds of protons in proteins, aspects that we will go into more detail for in the following passages of this Account.

2. THE PROTON AS A DETECTION NUCLEUS Apart from the narrow proton line widths, the reduction of the effective proton−proton dipolar couplings has multiple other benefits. On one hand, high-power proton decoupling is not necessary, which reduces the radio frequency energy irradiated into the sample in a given pulse sequence.11 Consequently, the number of scans per time can be drastically increased, at least to the point that it becomes limited by the proton T1 time. In fact, the interscan delay can be further reduced to a few hundred B


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Figure 2. Different aspects of proton-detected NMR. (A) Pulse sequences based on CP (blue) and INEPT (red) magnetization transfers as well as (B) the corresponding chicken α-spectrin SH3 spectra in black and red.29 (C) SH3 spectrum obtained after 1d HD-exchange (green) and reference spectrum (black).31 (D) Doping of protein microcrystals with Cu-EDTA and (E) concentration-dependent reduction of 1H T1 in 10% 1H-backexchanged, 2H SH3 by Cu-EDTA.11 (F) Assigned H/N correlation spectrum of a 29 kDa hCAII.26

protein spun at 100 kHz by Penzel et al.39) Figure 3A and C displays some of those experiments, here employed as purely dipolar versions for assignments of hCAII. These experiments have also been expanded to four dimensions by us and others.22,40−42 More sophisticated magnetization transfer pathways enable correlation between one amide group and either one (3D) or both shifts (4D) of the next one (Figure 3A (fourth scheme) and D).43 At around 60 kHz MAS, the exclusively dipolar transfer pathway of this hNcacoNH (or HNcacoNH) is highly efficient and yields better sensitivity than an hcaCBcaNH under comparable conditions. Moreover, the dispersion of 15N chemical shifts (spectral range divided by the effective line width) is the best among all backbone nuclei,43 such that the 3D hNcacoNH and 4D HNcacoNH yield exceptional ease and fidelity for assignment. High sensitivity, increased T2 times and low rf duty of these sequences facilitate higher dimensionality, which helps assigning large proteins and obtaining unambiguous distance restraints for structure calculation (see below). Obtaining carbon side chain shifts of deuterated samples is highly beneficial for structure, dynamics, and interactionsbut challenging. 1H-Detected scalar-transfer-based assignment strategies, employing side chain methyls for detection and TOBSY mixing,35 were first suggested by Agarwal and Reif.44 By extending this concept, we introduced 3D side chain-tobackbone experiments similar to C(C)NH-TOCSY spectra in solution (Figure 3A (bottom) and E).45,46 These yield carbon shifts for the complete side chain spin systems in one dimension with amide N/H dispersion in the other two dimensions. We implemented different versions, using either TOBSY (at 20 kHz MAS),45 MOCCA (at 27 and 60 kHz

MAS),46,47 or composite MOCCA mixing.26 The efficiency of the composite MOCCA variant, mixing aliphatics with aromatics and carbonyls, is shown in Figure 3E, obtained for hCAII within 2 days of measurement time (20% nonuniform sampling).26 With perdeuteration, side chain carbons are hard to reach via CP from amide protons. We created a scheme for a dual (and as such for effective) carbon excitation by combining both long-range H/C CP and 13C Boltzmann polarization (“COPORADE”, Figure 3B).45 Aliphatic proton shifts in protonated samples can be correlated with the backbone by using an HBHAcbcaNH experiment22 or via inverse second-order cross-polarization (iSOCP), spreading amide nitrogen magnetization to various (distant) protons via third-spin assisted polarization transfer.19

4. CHARACTERIZATION OF DISTANCES Characterization of protein structure constitutes one of the main solid-state NMR foci. Distance restraints can be obtained by probing through-space dipolar interactions between electron or/and nuclear spins via recoupling approaches or paramagnetic relaxation enhancements (PRE) (Figure 4). Among nuclear spins, protons have the strongest dipolar interactions and thus they yield the longest-range restraints. Direct proton−proton polarization transfer can be used for distance measurements. For proton-detected techniques, RFDR and DREAM transfer have been used to substitute the traditional spin-diffusion-based methods.9,14,16,48−52 When proton shifts are accessible, direct shift encoding of spatial proximity is straightforward and yields high sensitivity.14,48,49,53 The classic solution NMR approach, the 3D 15N-edited NOESY, was translated to the solid state by Zhou et al.48 We C


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Figure 3. Backbone and side chain assignment experiments based on proton detection. (A) Magnetization transfer schemes for 13C-based sequential assignments, amide-to-amide correlations, and side chain-to-backbone experiments. (B) Dual excitation of side chain carbons. (C) Example for a 13 C-based backbone walk: hCANH/hCAcoNH. (D) Strips of an amide-to-amide correlation (here: 3D hNcacoNH). (E) Side chain-to-backbone (S2B) correlation using composite-pulse MOCCA 13C−13C mixing. All experiments were recorded on DCN hCAII, 100% labile-proton-backexchanged, at 55.55 kHz MAS.26 Reproduced from ref 26. Copyright 2018 American Chemical Society.

Sampling54 and Iterative Soft Thresholding55 have yielded excellent spectra for HNNH experiments with only 2% sampling density.49 Low sampling density can be implemented easily in higher dimensionality, and higher sensitivity gains (by predominant sampling of the higher-intensity FIDs54) are obtained. (For practical information on NUS in the solid state and in solution the reader is directed to the extensive work of Hyberts and Wagner, Rovnyak and Hoch, Orekhov, or Freeman.55−57) Figure 4G and H shows a 2D slice extracted from an “ultrasparse NUS” 4D HNh-RFDR-hNH at diagonalpeak position K26.49 Generally, diagonal peaks in homonuclear mixing experiments are higher than any cross peak, easily overshadowing cross peaks with their peak envelopes, distorting the baseline, or creating wiggles in case of truncation. This is particularly severe for partially back-exchanged samples. In solids, a “diagonal-compensated” version, circumventing these problems, can be implemented with little s/n cost by subtraction of scans without mixing: Since diagonal peaks in

further expanded this approach to four dimensions, with which we could obtain well-resolved peaks, unambiguous contacts, and very long-range distance information.14 Similarly, the Meier group implemented a 4D HCCH DREAM mixing for distance information among methyl protons using CHD2-labeled methyls.53 Obviously, such experiments are always more beneficial in which both amides and methyls participate in through-space magnetization transfers simultaneously, yielding amide−amide, amide-methyl, and methyl−methyl distances. Thus, we developed a dual-CP-transfer pulse sequence element, with a proton rf field that fulfills the Hartmann−Hahn condition both with respect to a carbon as well as to a simultaneous 15N spin lock (see Figure 4D). For complex/ heterogeneous systems, both heteronuclei can be encoded in time-shared RFDR experiments, with shorter evolution times for 13C than for 15N for correct scaling; see Figure 4F.14 Four dimensional (4D) experiments are most versatile when recorded using nonuniform sampling (NUS). Poisson-Gap D


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Figure 4. Distance restraints from 1H-detected ssNMR approaches. (A) Amide attenuation (red) in N47C-MTSL-labeled SH3. (B) Probing spin− spin interactions using protons can involve nuclei and electrons. (C) Overlay of a reference H/N correlation (blue) with that of the MTSL-sample (red). Yellow shades denote attenuated peaks.58 (D) Prototypical pulse scheme of a time-shared correlation comprising simultaneous H/N and H/C CPs and a t1max-adjusted indirect heteronuclear dimension. (E) Structure of SH3 from automatically assigned time-shared 3D RFDR spectra using ARIA.14 (F) Time-shared H-RFDR-hNH (top strips) and hNh-RFDR-hNH (right strip) together with a time-shared H−N/C 2D reference.14 (G) Diagonal-compensated, ultrasparse NUS 4D HNh-RFDR-hNH, shown for K26 coordinates. A reference H/N correlation spectrum (in sky blue) is overlaid. (H) Identical, but without elimination of the diagonal.49 Panels (D)−(F) reproduced from ref 14. Copyright 2011 American Chemical Society. Panels (G) and (H) reproduced from ref 49. Copyright 2014 American Chemical Society. Panels (A) and (C) reproduced with permission from Wiley.

Figure 5. Proton detection in the framework of protein dynamics. (A) 15N R1ρ of deuterated and proton back-exchanged hCAII (PDB: 2CBA).26 (B) 15N relaxation dispersion data from multiangle, off-resonance R1ρ.64 (C) Differential relaxation rates for N−Hα and N−Hβ coherences.29 (D) CEST as an additional restraint in quantifying μs dynamics.64 (E,F) Theoretical 1H R1ρ relaxation rates close to rotational resonance conditions, differentiating 1H homonuclear and 1H/15N-heteronuclear dipolar contributions. (G) Simulated homonuclear dipolar relaxation contribution to 1H R1ρ rates as a function of motional time scales and order parameters (ω1/2π = 12 kHz, 40 kHz MAS).62 (H) Comparison of 1H and 15N R1ρ rates for SH3. Panel (C) reproduced from ref 29. Copyright 2010 American Chemical Society. Panels (E)−(G) reproduced from ref 62. Copyright 2017 American Chemical Society. Panels (B) and (D) reproduced with permission from Wiley. Panel (A) reproduced from ref 26. Copyright 2018 American Chemical Society.

C).58 However, for accurate distance restraints, it seems necessary to drastically dilute the MTSL-labeled samples with wild-type, deuterated proteins so that intermolecular PRE contributions become negligible.

a scan without mixing have a several-fold higher intensity than with the mixing on (owing to relaxation, pulse imperfections, and magnetization transfer away from diagonal), a single compensation scan can be subtracted from multiple ordinary scans to eliminate the diagonal (compare Figure 4G with H, respectively).49 Paramagnetic effects have been used very successfully for structure calculation.59 Effects as long as 30 Å can be induced by electron−proton paramagnetic relaxation enhancement (PRE). This was demonstrated for microcrystalline SH3 that was spin-labeled with MTSL at position N47C (Figure 4A and

5. ELUCIDATION OF PROTEIN DYNAMICS Those conditions which enable proton detection (fast spinning and/or extensive deuteration) also facilitate quantification of residue-specific motion occurring on the picosecond-to-millisecond time scale. For protonated samples at slow spinning, evolution of dipolar couplings is imperfectly suppressed by E


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Figure 6. 1H-detected approaches for heterogeneous samples. (A) H/N correlation of Tau PHFs.23 (B) H/N correlation of hydrophobin overlaid with a solution-state monomer spectrum. (C) 4D NUS HNh-RFDR-hNH correlations on hydrophobin for distance restraints. (D) Excerpt of the 4D backbone walk from 4D NUS hCACONH and hCOCANH.22 (Upper right: Strip from a 3D hNcacoNH.) (E) Negative-stain EM image of Tau PHFs and (F) of hydrophobin rodlets.71 (G) Magnetization transfer in the 4D experiment shown in (D). Panel (A) reproduced from ref 23. Copyright 2017 American Chemical Society. Panels (B) and (F) reproduced with permission from Wiley. Panel (G) reproduced from ref 26. Copyright 2018 American Chemical Society.

high sensitivity using 2D H/N experiments. For complex targets (peak overlap in 2D H/N correlations), pseudo-4D readout of relaxation rates via 3D data is straightforward.26 As such, characterization of large proteins is feasible; Figure 5A demonstrates individual R1ρ rates for the 260-residue hCAII, assessed via consecutive 15N relaxation-edited 3D hCANH spectra.26 Consequently, ssNMR can quantify the motional parameters not only for those proteins that are not soluble but increasingly also for those that are simply too large for solution NMR techniques. With fast spinning and/or deuteration, almost any solutionstate NMR approach for assessment of dynamics can be rendered for solid-state NMR. Most suitably, given its negligible influence by same-spin neighbors, 15N R1ρ can be obtained in various ways, e.g., via a multiangle off-resonance assessment, see Figure 5B. Figure 5C shows differential relaxation, a parameter previously limited to solution NMR.29,30 R1 and R2 can report on picosecond−nanosecond and microsecond−millisecond motion, respectively; hetNOE is insensitive and would give the same information as R1.10 Chemical exchange saturation transfer (CEST) profiles can be used to add information to relaxation dispersion and thus improve the reliability of conformational exchange data (see Figure 5D).64 Generally, relaxation dispersion studies are very

sample rotation (due to second-order terms of the Magnus expansion in Average Hamiltonian Theory), leading to dipolar dephasing and spin diffusion, which inseparably add to the relaxation rates of interest from stochastic local motion.10 For a fully protonated sample, at least 40 kHz spinning frequency should be applied to quantify 15N relaxation.60 For slower (∼20−30 kHz) spinning, deuteration is necessary to probe parameters like R1, R1ρ, or 1H−15N cross-correlated relaxation. Under fast MAS and/or extensive deuteration, also protons can be probed for assessing dynamics. This allows access to different motional modes and has certain practical advantages and disadvantages over 15N-based dynamic studies due to differences in the origin of relaxation.61,62 Undesired contributions, however, scale with the interaction strength, influenced by the gyromagnetic ratio.10 Accordingly, faster spinning and extensive deuteration are required for 13C (also necessitating 12C dilution)63 or 1H relaxation studies. With paramagnetic doping using Cu(II) (see above),11,27 R1 rates will be partially influenced. However, due to electronic relaxation rates typically in the nanosecond regime, R2 (or R1ρ) rates, including relaxation dispersion or relaxation rotation interference (see below), are found to be largely unaffected.11 Practically, with sharp proton line widths, proton-detected residue-specific 15N relaxation can be assessed easily and with F


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Figure 7. Outlook on proton usage in the absence of deuteration at ultrafast MAS. (A) H/N correlation of hCAII at 111 kHz, with a picture of its crystal structure (PDB code 2CBA). (B) H/C correlation of hCAII at 111 kHz. In the 2D spectra, peak overlap is observed particularly for aliphatic H/C correlation peaks. (C) Rotor size of a 0.7 mm rotor in comparison to a match and paper clip. (D) Side chain carbon assignment of selected hCAII residues based on a 3D hCCH walk.

versatile, where R1ρ-based relaxation dispersion is preferred over CPMG due to the better suppression of coherent effects. Also, due to the interference of the spinning speed and irradiation field strength of an R1ρ measurement, microsecond time scale motion can be explored in a unique fashion.62,65 Figure 5 E and F shows theoretical profiles for rotation relaxation interference for homonuclear and heteronuclear dipolar contributions. Figure 5G shows 1H R1ρ dependence (using regular, i.e., far-from-rotary-resonance conditions) on the motional correlation time.62 Figure 5H compares regular 1 H and 15N relaxation rates for the SH3 domain. Alternatively, direct dipolar couplings (or CSA) can be measured and the motion-related time-averaged order parameter deduced from their values, even at fast MAS.66

6G) provide intra- and interresidual correlations between amides and backbone carbons.22,40 With proton line widths being dominated by inhomogeneous contributions and using highly efficient homonuclear dipolar transfers, a single fully protonated preparation spun “only” at 56 kHz yielded acceptable spectra for comprehensive backbone assignments. In such cases, deuteration would hardly improve resolution, rather one can exploit the high proton abundance for increased initial magnetization. Generally, similar assignment approaches (higher-dimensional spectra, NUS, and use of side chain shifts) as for high-molecular-weight microcrystalline proteins can be used for heterogeneous samples. For reliable assignments of Tau, we also recorded a 3D hNcacoNH experiment (upper right corner in Figure 6D), and a purely dipolar-based 3D hCBCANH tailored for protonated samples at 56 kHz. These experiments yielded more comprehensive and reliable assignments of Tau PHFs than previous 13C-based techniques with only 1 mg of protein,22 allowing comparative studies on the molecular origin of Tau polymorphism.23 (See a negative-stain electron micrograph in Figure 6E.) For distance restraints in a deuterated and 100% backexchanged sample of the functional amyloid hydrophobin (Figure 6F), we used again a 4D HNh-RFDR-hNH. Despite an equally discouraging hydrophobin H/N plane (Figure 6B) as for Tau, we could obtain a number of unambiguous and ambiguous distance restraints (Figure 6C). This is a first step toward determination of the elusive three-dimensional rodlet fold.

6. HETEROGENEOUS SAMPLES Many (potential) solid-state NMR targets comprise significant structural inhomogeneity, as in the case of disease-related and functional amyloids. Often only intensive sample optimization to reduce heterogeneity could make ssNMR viable,67,68 involving seeding procedures69 or truncation of the primary sequence to a better-defined core region. Only few studies in the literature have employed proton detection for heterogeneous samples yet. As can be seen from the heterogeneous line shapes for resolved resonances in Aβ fibrils,70 heterogeneous line broadening is particularly severe for amide protons, whose shifts are sensitive to distortions of hydrogen bond geometries. Nevertheless, the sensitivity gain of proton detection can improve assignment and structure calculation when combined with additional heteronuclear dimensions. We used fourdimensional assignment strategies via a proton-detected sequential walk based on 13C shifts to assign the largely heterogeneous Tau paired helical filaments (PHFs, see an assigned H/N correlation in Figure 6A and part of the 4D backbone walk in Figure 6D). The two complementary 4D NUS experiments (hCACONH and hCOCANH, see Figure

7. CONCLUSIONS AND OUTLOOK Proton detection in ssNMR has developed from an eccentric niche to a serious alternative to carbon detection, including assignments, structure calculation, assessment of dynamics, and interactions. Its success is proof that tedious developments need to be pursued as a joint effort of academic science and industry. G


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Accounts of Chemical Research Faster spinning decreases the homogeneous proton line width, relieving the need for deuteration. Figure 7A and B shows H/N and H/C correlations of fully protonated hCAII at 111 kHz MAS. Important arguments for 0.7 mm and smaller rotors (Figure 7C) are that (i) deuteration is not possible for many proteins of current scientific interest even for minimal protein quantities and (ii) quantitative back-exchange of amide protons often necessitates unfolding and refolding, a serious hurdle for many proteins. Despite higher homogeneous line widths compared to amides even at 111 kHz,21 aliphatic protons will be advantageous due to their insusceptibility to variations in amide bond geometry that amide protons suffer from. (See an Hali-detected hCCH-correlation in Figure 7D.) Apart from sequential assignment, we expect that structure calculation and dynamics will benefit from the possibilities arising with increasing spinning frequencies. Hopefully, our methodological developments presented here will help to keep on improving proton detection and stimulate others to join in engineering versatile strategies.

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chemistry in Göttingen, he was appointed Associate Professor at the Ludwig-Maximilians-University in Munich. His research is focused on development and application of new NMR methodology.

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ACKNOWLEDGMENTS

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REFERENCES

This research was supported by the Deutsche Forschungsgemeinschaft (SFBs 803, TP 04, and 749, TP A13, as well as the Emmy Noether program), the Verband der Chemischen Industrie (VCI) in terms of a Liebig junior group fellowship, the Australian Research Council (Discovery Early Career Research Award), the Excellence Cluster CiPS-M, and the Center for NanoScience (CeNS). We would like to thank Dr. Himanshu Singh for critical reading of the manuscript and Dr. Frank Engelke, Bruker, for his kind information on coil sensitivity. R.L. would like to acknowledge Dr. Paul Schanda for supportive discussions and is very grateful to Prof. Bernd Reif, with whom the journey into employing protons for solid-state NMR started.

AUTHOR INFORMATION

Corresponding Author

(1) Paulson, E. K.; Morcombe, C. R.; Gaponenko, V.; Dancheck, B.; Byrd, R. A.; Zilm, K. W. Sensitive High Resolution Inverse Detection NMR Spectroscopy of Proteins in the Solid State. J. Am. Chem. Soc. 2003, 125, 15831−15836. (2) Chevelkov, V.; Rehbein, K.; Diehl, A.; Reif, B. Ultra-High Resolution in Proton Solid-State NMR Spectroscopy at High Levels of Deuteration. Angew. Chem., Int. Ed. 2006, 45, 3878−3881. (3) Chevelkov, V.; van Rossum, B. J.; Castellani, F.; Rehbein, K.; Diehl, A.; Hohwy, M.; Steuernagel, S.; Engelke, F.; Oschkinat, H.; Reif, B. 1H Detection in MAS Solid-State NMR Spectroscopy of Biomacromolecules Employing Pulsed Field Gradients for Residual Solvent Suppression. J. Am. Chem. Soc. 2003, 125, 7788−7789. (4) Ishii, Y.; Tycko, R. Sensitivity Enhancement in Solid State 15N NMR by Indirect Detection with High-Speed Magic Angle Spinning. J. Magn. Reson. 2000, 142, 199−204. (5) Ishii, Y.; Yesinowski, J. P.; Tycko, R. Sensitivity Enhancement in Solid-State C-13 NMR of Synthetic Polymers and Biopolymers by H-1 NMR Detection with High-Speed Magic Angle Spinning. J. Am. Chem. Soc. 2001, 123, 2921−2922. (6) Zhou, D. H.; Shah, G.; Cormos, M.; Mullen, C.; Sandoz, D.; Rienstra, C. M. Proton-Detected Solid-State NMR Spectroscopy of Fully Protonated Proteins at 40 kHz Magic-Angle Spinning. J. Am. Chem. Soc. 2007, 129, 11791−11801. (7) Lewandowski, J. R.; Dumez, J.-N.; Akbey, Ü .; Lange, S.; Emsley, L.; Oschkinat, H. Enhanced Resolution and Coherence Lifetimes in the Solid-State NMR Spectroscopy of Perdeuterated Proteins under Ultrafast Magic-Angle Spinning. J. Phys. Chem. Lett. 2011, 2, 2205− 2211. (8) Huber, M.; Böckmann, A.; Hiller, S.; Meier, B. H. 4D Solid-State NMR for Protein Structure Determination. Phys. Chem. Chem. Phys. 2012, 14, 5239−5246. (9) Andreas, L. B.; Jaudzems, K.; Stanek, J.; Lalli, D.; Bertarello, A.; Le Marchand, T.; Cala-De Paepe, D.; Kotelovica, S.; Akopjana, I.; Knott, B.; Wegner, S.; Engelke, F.; Lesage, A.; Emsley, L.; Tars, K.; Herrmann, T.; Pintacuda, G. Structure of Fully Protonated Proteins by Proton-Detected Magic-Angle Spinning NMR. Proc. Natl. Acad. Sci. U. S. A. 2016, 113, 9187−9192. (10) Schanda, P.; Ernst, M. Studying Dynamics by Magic-Angle Spinning Solid-State NMR Spectroscopy: Principles and Applications to Biomolecules. Prog. Nucl. Magn. Reson. Spectrosc. 2016, 96, 1−46. (11) Linser, R.; Chevelkov, V.; Diehl, A.; Reif, B. Sensitivity Enhancement Using Paramagnetic Relaxation in MAS Solid-State NMR of Perdeuterated Proteins. J. Magn. Reson. 2007, 189, 209−216. (12) Linser, R.; Fink, U.; Reif, B. Proton-Detected Scalar Coupling Based Assignment Strategies in MAS Solid-State NMR Spectroscopy Applied to Perdeuterated Proteins. J. Magn. Reson. 2008, 193, 89−93.

*E-mail: [email protected]. ORCID

Petra Rovó: 0000-0001-8729-7326 Rasmus Linser: 0000-0001-8983-2935 Author Contributions †

S.K.V. and P.R. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. Biographies Suresh K. Vasa obtained his master’s degree in physics from the Indian Institute of Science Bangalore. He received his Ph.D. from Radboud University Nijmegen in 2013 for his work on microMAS NMR in the group of Arno Kentgens. Later he worked as a postdoctoral fellow in Adam Lange’s group at the Max Planck Institute for Biophysical Chemistry in Göttingen. Currently, he is a postdoc with Rasmus Linser at the Ludwig-Maximilians-University in Munich. His research includes NMR methodology developments and their applications for challenging biomolecular systems. Petra Rovó received her M.Sc degree in chemistry from the Eötvös Loránd University Budapest in 2009 and her Ph.D. from the same institute in 2014. During her Ph.D., she was a Fulbright fellow at University of Notre Dame working with Jeffrey W. Peng. She joined Rasmus Linser’s group at the Max Planck Institute for Biophysical Chemistry in Göttingen in 2014. Currently, she is a postdoc in the Linser group at the Ludwig-Maximilians-University in Munich. Her research interest is solid-state NMR relaxation and protein dynamics. Rasmus Linser studied chemistry at the University Göttingen, Germany, and Universidad Autónoma de Madrid, Spain. He obtained his Ph.D. in the group of Bernd Reif at the FMP in Berlin in 2010. After postdoctoral work at the Analytical Centre of the University of New South Wales (UNSW), Sydney, Australia, he held a Discovery Early Career Research Award-financed dual appointment at UNSW and Harvard Medical School, Boston, MA, in the lab of Gerhard Wagner. After 2 years as a Liebig- (VCI) and Emmy Noether (DFG)funded junior group leader at the Max Planck Institute for biophysical H


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Protons as Versatile Reporters in Solid-State NMR Spectroscopy

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