Structure and backbone dynamics of a microcrystalline metalloprotein by solid-state NMR

Abstract

We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with ¹H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of ¹⁵N and ¹³C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu⁺ (diamagnetic) or Cu²⁺ (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to ¹H-¹H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a Gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.

Structure determination of proteins plays a central role in understanding key events in biology. Although the structure of many proteins can be obtained from single-crystal X-ray diffraction, or by solution-state nuclear magnetic resonance (NMR) spectroscopy, there is nevertheless a range of important substrates for which structures cannot be determined today. These include immobile systems lacking long-range order such as protein aggregates, large complexes and membrane-bound systems.

Solid-state NMR has the unique potential to study, with atomic resolution, systems of this nature, and spectacular progress has been made in this area over the last decade (1). There are today a small handful of structures obtained by solid-state NMR, from microcrystalline samples to fibrils and membrane-associated systems (2). Additionally, solid-state NMR is uniquely sensitive to site-specific protein dynamics over a broad range of timescales, and a number of demonstration studies have recently appeared for model proteins (3).

The use of perdeuterated proteins has very recently opened the way to highly sensitive proton-detected solid-state experiments (4). Despite early proof-of-principle papers (5), this approach only became popular with the realization that amide sites must be only partially reprotonated (typically 10–30% back exchange) (6–8) to yield well-resolved ¹H spectra. This represented a significant compromise in sensitivity to gain resolution, and effectively made the determination of internuclear distances impractical, with few exceptions (9–11). We have recently shown how this problem can be completely overcome by using 100% reprotonation of exchangeable sites, without loss of resolution, if perdeuteration is combined with ultrafast MAS (60 kHz) (12, 13). Well-resolved “fingerprint” spectra may then be acquired rapidly, opening up the way to the detection of a range of structurally important parameters.

Paramagnetic effects have long been used and today play a central role in solution-state NMR structural determinations in metalloproteins (14–16), or in biomolecules covalently modified with a spin-label (17–19). Importantly, paramagnetic effects can manifest themselves as either shifts (contact or “pseudocontact” shifts) or enhanced relaxation (PREs), depending on the character of the metal center. These effects act up to very long distances, and carry a well-defined dependence on the nuclear position with respect to the paramagnetic center. In solids, measuring pseudocontact shifts is relatively straightforward, and when they occur, they can be used as invaluable structural constraints (20–22). However, measuring PREs in solids is a prime example where traditional detection methods have difficulty to provide sufficient sensitivity. While enhanced relaxation caused by a paramagnetic center can be exploited for fast recycling and condensed data collection approaches relying on ¹³C detection (23–25), the addition of paramagnetic dopants is not appropriate for the quantitative measurement of relaxation times as a source of structural and dynamic information. As a result, site-specific PREs in the solid state have only been reported on one small model protein (GB1) by the use of cysteine-containing mutants with paramagnetic tags attached (26, 27).

Here we demonstrate a new approach to improving structural and dynamical determination of large metalloproteins. Using ¹H-detected multidimensional experiments at high magnetic field under ultra-fast magic-angle spinning (MAS), combined with perdeuteration, we can efficiently measure a large number of ¹³C and ¹⁵N nuclear relaxation rates with high sensitivity in a large metalloprotein as well as ¹H-¹H distance restraints. This leads to the complete description of its site-specific motions in the ns-us scale and to the evaluation of a large number of long-range paramagnetic restraints for the determination of the full structure of the molecule, including a well-defined active site.

We apply these methods to the enzyme superoxide dismutase (SOD) (28, 29), which coordinates a Cu and a Zn ion and has the physiological function of protecting cells from oxidative stress by catalyzing the dismutation of the superoxide anion. The Cu ion is essential to catalysis, and cycles between the (paramagnetic) Cu²⁺ state and (diamagnetic) Cu⁺ state during the reaction.

PREs are obtained from the difference in rates, and when added to ¹H-¹H distance restraints, are shown to improve the precision of the structure by a factor of two. Site-specific order parameters and timescales of motion are obtained by a Gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecules.

Results and Discussion

NMR Experiments.

Fig. 1 shows ¹H-detected ¹⁵N-¹H correlation spectra (¹⁵N-¹H Cross-Polarization Heteronuclear Single Quantum Coherence or CP-HSQC, Fig. S1) of microcrystalline SOD in its Cu⁺,Zn²⁺ state (A-B, diamagnetic) and Cu²⁺,Zn²⁺ state (C, paramagnetic) recorded using ultrafast MAS at 60 kHz at 850 MHz.

Fig. 1.

Solid-state ¹⁵N-¹H CP-HSQC spectra of SOD, recorded at 850 MHz ¹H Larmor frequency and using 60 kHz MAS. (A) Cu⁺,Zn²⁺-SOD. (B) and (C) show close-up regions of CP-HSQC spectra of Cu⁺,Zn²⁺-SOD and Cu²⁺,Zn²⁺-SOD, respectively.

Samples are U-[²H,¹³C,¹⁵N] and have been allowed to exchange with H₂O after expression, such that all exchangeable sites are protonated (100% back-protonation). As shown recently in the case of the analog Zn²⁺-SOD, this protocol yields here excellent resolution and sensitivity with ¹H coherence lifetimes of generally around 10 ms with line widths of around 100 Hz (13). These spectra can be acquired in 15 min using only 3.5 mg (< 0.22 μmol) of protein in a 1.3 mm rotor.

In the case of Cu⁺,Zn²⁺-SOD (which is diamagnetic), 136 backbone amide resonances out of 147 nonproline residues were assigned using ¹H-detected triple-resonance 3D experiments, as detailed in the SI Text. In the case of Cu²⁺,Zn²⁺-SOD, 116 backbone amide resonances were assigned. In this latter case, we expect that nuclei particularly close to the Cu²⁺ ion are relaxed very rapidly and thus evade detection. Details of resonance assignment procedures are given in the SI Text.

103 resonances were resolved in the CP-HSQC, indicating that the CP-HSQC experiment is an efficient detection block for ¹⁵N or ¹³C relaxation measurements. Thus, the ¹⁵N-¹H CP-HSQC dipolar correlation module was combined with a ¹⁵N inversion-recovery block (30, 31), ¹⁵N spin-lock (32, 33), or additional ¹³C-¹⁵N specific transfers (34) and ¹³C inversion-recovery (35) into a new set of extremely sensitive and resolved experiments. Pulse schemes are shown in Fig. S1.

Using this approach, we have measured ¹⁵N R₁, ¹⁵N R_1ρ as well as ¹³CO R₁ in the Cu⁺,Zn²⁺, Cu²⁺,Zn²⁺-SOD samples. We note that ultrafast MAS has an added advantage in relaxation studies because the proton bath is well-decoupled, alleviating potential interfering effects of coherent contributions. This is particularly relevant in the site-specific measurement of the ¹⁵N R_1ρ and ¹³CO R₁, which are both particularly susceptible to residual effects. It has been shown that these parameters can be accurately measured under MAS by using MAS frequencies > 40 kHz and a spin-lock nutation frequency > 10 kHz (31, 33, 35).

Examples of the decay curves are shown in Fig. S2, and the complete set of relaxation rates obtained for both Cu oxidation states is plotted in Fig. 2, and listed in Table S1. In the case of Cu⁺,Zn²⁺-SOD, ¹⁵N R₁s were generally in the range 0.01–0.05 s^-1, with faster R₁s generally in loop regions. In the case of ¹³CO R₁, most fell in the range 0.1–0.5 s^-1. In the case of ¹⁵N R_1ρ, which is dependent upon slower frequencies of motion than R₁, rates were 2–10 s^-1 typically.

Fig. 2.

Relaxation rates for microcrystalline SOD. (A) ¹⁵N R₁ for Cu⁺, Zn-SOD, (B) ¹⁵N R₁ for Cu²⁺, Zn-SOD, (C) ¹⁵N R_1ρ for Cu⁺, Zn-SOD, (D) ¹⁵N R_1ρ for Cu²⁺, Zn-SOD, (E) ¹³CO R₁ for Cu⁺, Zn-SOD, (F) ¹³CO R₁ for Cu²⁺, Zn-SOD. The secondary structure is indicated above the figure, showing the positions of the Cu-coordinating histidine residues with asterisks. The areas in gray boxes are those for which the residue comes within 12 Å of the Cu²⁺ ion according to the single-crystal X-ray structure (PDB code: 1SOS).

In the case of the paramagnetic Cu²⁺,Zn²⁺-SOD sample, substantial increases in ¹⁵N R₁ and ¹³CO R₁ were observed for certain sites, whilst others remained consistent with the diamagnetic form. These differences are due to the PRE induced by the Cu²⁺ ion by the Solomon mechanism (36), and will be strongest for residues close to the metal center. The ¹⁵N R_1ρ is affected by large diamagnetic contributions (notably influenced by fluctuations in the μs-ms time scale). PREs are dominated by electron fluctuation in the ns range, and thus influence R_1ρ less than R₁. The ¹⁵N R_1ρ in the Cu²⁺ and Cu⁺ oxidation states are therefore quite similar, as seen in Fig. 2.

Paramagnetic Relaxation Enhancements (PREs).

In a paramagnetic system, nuclear relaxation is made faster due to interactions between the unpaired electron(s) of the paramagnetic center and the nuclear spins (36). PREs can thus be measured in the case that paramagnetic and diamagnetic forms of the sample are available. PREs were obtained here by subtracting the diamagnetic spin relaxation rates from those of the paramagnetic sample (Fig. S2 G and H), and translated into distances from the metal center using the well-known relationship (SI Text) and a Cu²⁺ electronic correlation time of 2.5 ns, based on literature values (36, 37). These ¹⁵N R₁ and ¹³CO R₁ PREs quantify distances as close as 10 Å from the Cu ion and as far as 20 Å (Table S2). Contributions from intermolecular effects (38) were neglected. (this approximation was subsequently validated and shown to have no impact on the analysis for this large protein).

In order to use these data as structural restraints, PREs were incorporated into a structure determination protocol in combination with ¹H-¹H distance restraints, chemical shift-derived dihedral angle restraints and ambiguous H-bond restraints. For each distance predicted from the PRE data we ascribed to the corresponding nucleus an upper distance limit to the Cu ion 3 Å greater than the predicted distance, and a lower limit 3 Å less than the predicted distance. In total, we made use of 85 ¹³C PREs and 90 ¹⁵N PREs. In addition there were 25 ¹H-¹⁵N cross-peaks observable in the diamagnetic but not paramagnetic form. In these cases, it was assumed that this was due to broadening of the ¹H resonances beyond detection, and an upper distance limit to the Cu ion of 10 Å was used, without lower limit (39). The remaining residues corresponded to peaks for which accurate PRE determination was not possible.

¹H-¹H distance restraints were measured through a 3D (H)NHH_RFDR experiment (13, 40), to which we applied the automatic assignment and structure calculation programs ATNOS/CANDID (41, 42), implemented in UNIO, to iteratively assign the (H)NHH_RFDR and obtain a fold. This process (13) uses cycles of concerted peak assignment and structure calculation. In the first cycle (but not in subsequent cycles), the homologous Cu²⁺,Zn²⁺-SOD crystal structure (PDB code 1SOS (29)) was used to identify artifacts that might be mistaken for peaks in the spectrum not consistent with any realistic assignment, and to ensure that assignments consistent with the homologous structure were not erroneously discarded. However, assignments of ¹H-¹H contact peaks were made independently of the homologous structure in all cycles. Details are found in the methods section.

A total of 257 unique and nontrivial proton-proton distance restraints were obtained from the (H)NHH_RFDR experiment performed on Cu⁺,Zn²⁺-SOD. Of these, 99 were between residues both in beta-strands, 63 between a beta-strand residue and a loop residue, and 95 between residues both in a loop (Fig. S3 A and B). PRE-derived distance restraints were then added with no further assignment procedure and the structure re-calculated. In cases where the PRE-derived restraints were consistent violations (violations in more than half the members of the final ensemble), the distance bounds for these restraints were made wider and the calculation repeated. The final bundles of 20 structures, with and without PRE-derived restraints, are shown in Fig. 3. The structure is composed of a well-defined beta-barrel, two short alpha helices, and long loop regions containing the histidines coordinating the Cu^+/2+ and Zn²⁺ ions. No experimental constraints other than PREs were used to determine the position of the Cu^+/2+ ion in the molecule.

Fig. 3.

The structure of SOD obtained from solid-state NMR, showing 20-structure bundles overlaid by backbone heavy atoms. A and C structure obtained using automatically assigned distance restraints from a (H)NHH_RFDR spectrum and chemical-shift derived dihedral angle restraints, viewed from two different angles. (B and D) The same restraints used as in A and C but including R₁ PREs from ¹⁵N and ¹³CO. The Cu ion is shown as a red sphere.

The qualitative improvement in the structure by the inclusion of PRE-derived restraints is immediately evident in this figure, and is indicated quantitatively by the drop in backbone-heavy atom RMSD from 2.90 Å without PREs to 1.66 Å with PREs. In the beta-barrel, the RMSD dropped from 1.54 Å without PREs to 1.01 Å with PREs, whilse in the loops the RMSD dropped from 3.42 Å to 1.91 Å (Fig. S3C). Indeed, the backbone structure of the loop regions in the vicinity of the Cu ion is well defined only when PREs are used, despite quantitative PRE-derived restraints being available only for regions at least 10 Å distal to the Cu ion. The Cu ion itself, with the inclusion of PRE-derived restraints, is positioned with an RMSD of 0.334 Å when the structure bundle is superimposed using all backbone heavy atoms. The use of PRE-derived restraints has therefore enabled a much more detailed characterization of the active site of the enzyme than in their absence. In particular, a lack of information around the Cu ion has been overcome by multiple long-range restraints. We note that, with only a single paramagnetic centre, in the absence of the short-range RFDR-derived ¹H-¹H distance restraints, we did not obtain a well-defined structure.

In order to validate the implementation of PREs, Fig. 4 shows the correlation between the PRE-derived distances from the microcrystalline powder and the distances in the determined structure (a comparison with distances obtained from single-crystal X-ray diffraction on Cu²⁺,Zn²⁺-SOD is displayed in Fig. S3 D and E). The agreement is generally good, and the deviation between the PRE-derived distances and NMR structure distances is nearly always less than 2.5 Å. This vindicates the upper and lower distance restraints of 3 Å above and below the predicted distances respectively in the structure refinement. Those for which an adjustment to the upper and lower bounds was applied (on account of being violations) are shown in gray in Fig. 4. These outliers are generally attributable to low signal intensity, or the peak being partly overlapped.

Fig. 4.

(A and B) Agreement between Cu-nucleus distance in the NMR structure bundle and the PRE-derived distances for (A) ¹⁵N and (B) ¹³CO. (C) The PRE-derived distances (blue lines) plotted on the X-ray crystal structure on which secondary structure elements are labeled. The Cu ion is shown in red. In all graphs, the dotted lines indicate the 2.5 Å deviation from perfect agreement. (D) The PRE-inclusive NMR structure (blue) overlaid with the X-ray single-crystal structure (PDB code 1SOS, magenta). In A and B the unfilled gray points are those identified as violations in the structure calculation, and for which wider distance bounds were applied than the ± 3 Å bounds used generally.

Making use of data acquired on the Cu⁺ and Cu²⁺-bound states assumes that the structural variations upon change in the oxidation state are minor, as established by several studies of globular redox proteins in solution (43). In this case, comparison of shifts (Fig. S4) shows that the two structures are very similar. PREs for nuclei in sufficient proximity to the Cu ion to be sensitive to any major structural differences between the two states were not measured, because they are rendered unobservable by large transverse PREs, justifying the use of a point-dipole approximation for the paramagnetic effects, validating the method further.

In summary, we find that in concert RFDR and PRE-derived distance restraints (Fig. 4C) are able to determine the structure with good precision (Fig. 3 B and D) and good accuracy (Fig. 4D), as shown by the comparison with the X-ray single crystal structure (PDB code: 1SOS).

Site-Specific Backbone Dynamics.

Because the PRE method involves measuring relaxation times in the diamagnetic form, a natural extension of the protocol is to determine dynamics in concert with the PRE-restrained structure.

Nuclear spin relaxation parameters determined by NMR spectroscopy are powerful and widely used reporters of dynamical information in studies of proteins, in which structural flexibility is understood to be highly important (44). While solution-state NMR methods to study dynamics are today very sophisticated, using up to 30 dynamical restraints per residue (45), extensive site-specific relaxation studies of dynamics in solid proteins are rare (31, 33). Despite the technical obstacles, solid-state nuclear relaxation times should be exquisitely sensitive to functionally relevant internal motions. These motions are generally preserved in crystals, while overall rotational diffusion, which dominates relaxation in the solution state, is absent. Because relaxation is determined by motion, not static disorder, the information provided is inherently different from crystallographic B-factors.

In the case of the (diamagnetic) Cu⁺,Zn²⁺-SOD, we have performed a determination of the backbone dynamics from the site-specific set of ¹⁵N R₁ and R_1ρ according to the recently demonstrated GAF approach (33). To calculate relaxation decays, we assume that ¹⁵N relaxation is dominated by its CSA and the dipolar coupling with the directly bonded proton (32, 33). To describe the dynamics, we have used the 1D GAF model (46, 47), for which we assume that the NH bond vector undergoes diffusive fluctuations in the peptide plane subject to a harmonic potential. In this model, dynamics are specified by the GAF diffusion timescale and an order parameter related to the variance (equivalently the angular restriction) of the GAF motions. The relevant formulae can be found in the Methods section in the SI Text.

In Fig. 5, the order parameters and timescales determined for the 1D GAF model are plotted along the amino acid sequence, and depicted on the dimeric structure of SOD. In this representation we explicitly assume that every copy of the monomers comprising each SOD dimer undergo identical motions. Our measurements inherently detect the average dynamics. Dynamics parameters are tablulated in Table S3, those obtained with different motional models are plotted in Fig. S5, and the form of the GAF correlation function is depicted in Fig. S6.

Fig. 5.

Internal dynamics of microcrystalline Cu⁺, Zn-SOD as determined by the 1D Gaussian axial fluctuation (1D GAF model). (A) Order parameters, (B) GAF timescales, (C) effective timescales of internal motion representing the best mono-exponential approximation to the correlation function, equivalent to the Lipari-Szabo model-free effective correlation time, (D) order parameters projected onto the dimeric structure of SOD (PDB code: 1SOS). Larger width and more red color denote a lower order parameter. (E) GAF timescales projected onto the dimeric structure of SOD. Larger width and more turquoise color denote larger (slower) GAF timescales. In D and E, the Cu ion is shown as a gold sphere, and the Zn ion as a blue sphere.

It is immediately seen that the solid-state NMR order parameters are in qualitative agreement with general expectations of protein structure; high order parameters are seen in the beta-barrel, whereas lower order parameters are found in the loop regions. It is important to note that an order parameter is not affected by static disorder, but is instead determined entirely by dynamics. The low order parameters observed in loops therefore indicate that such regions are indeed flexible. The mean order parameter obtained was 0.92 and varies between 0.97 and 0.65 with a clear bias towards higher order parameters.

High order parameters were found for β-strands. Strands 4, 7, and 8, which constitute the core of the protein fold, have higher values. The residues directly involved in metal-protein interactions (H46, H48, H120 coordinating Cu⁺ and H63, H71, H80 and D83 coordinating Zn²⁺) feature also generally very high (mostly > 0.95) order parameters, indicating quite rigid binding sites. A notable exception is H63, which is involved in Cu²⁺ and Zn²⁺ coordination, but is not bound to Cu⁺ in the reduced state. Its relatively low order parameter (S² = 0.90, the lowest amongst the metal-coordinating residues) may thus be the result of increased motility in the absence of side-chain metal binding.

If we consider the deviation between the NMR and the single crystal X-ray structures, in some regions where RFDR restraints are sparse, the agreement can be poor, with the largest deviations occurring in the 22–24, 107–109, and 124–130 regions (Figs. 4D and 5). The region 124–130 has generally high order parameters, indicating an absence of flexibility, despite the indication of disorder from the PRE analysis, and the variability amongst the various molecules in the asymmetric units (29), and between several X-ray crystal structures (48, 49) in this region. As for residues 107–109, the order parameters are lower, suggesting that this region is dynamic even in the crystal, also consistent with the higher crystallographic B-factors.

There is considerable variation in the timescale of motion, from 11 ns to 956 ns. The mean timescale of motion T_GAF is 222 ns. This corresponds to a “model-free” effective correlation time T_EFF of 25 ns, a timescale that is particularly difficult to measure using solution-state NMR due to overall rotational diffusion, which occurs on a timescale of 25 ns for dimeric SOD (50). It is possible, of course, that multiple timescales of motion occur, or that collective motions are also present, methods for the evaluation of which exist (51). The evaluation of such models, however, is beyond the scope of the current study. In general, GAF timescales are higher in beta-strands than in loops, implying greater contributions of fast motions in loop regions relative to beta strands. In the beta-barrel, there is a clear separation of the timescale of motion of the “outer” sheet (strands 1, 2, 3, and 6) and those that form the “inner” sheet at the core of the molecule (strands 4, 5, 7, and 8). This observation may be indicative of concerted dynamics in each sheet, which has been suggested previously (SOD solution) but not quantified due to the difficulty of measuring this timescale by any other means. The high order parameters show that such motions are of low amplitude, but are detectable here nonetheless, such is the sensitivity of the technique. However, these measurements are not unambiguously diagnostic of concerted motions, and extensive further experimentation would be required to validate such models.

The three Cu⁺-coordinating residues have very consistent GAF timescales (H46: 491 ns, H48: 396 ns, H120: 435 ns), and all are well above (slower than) the mean timescale of 222 ns. H63, conversely, has a GAF timescale of 108 ns, which is one of the lowest (thus fastest) recorded. At the other extreme, D83, which is also involved in Zn coordination, shows the slowest motion of all recorded, with a GAF timescale of 956 ns. As for the order parameters, these timescales appear to correlate to the binding pattern in the active site.

A model-free analysis of the backbone dynamics of Cu⁺, Zn-SOD has also been published using solution NMR (50). On the whole, the solution-state order parameters are in good agreement with those obtained for our microcrystalline sample, such that the amplitude of dynamics may be similar between the two states. A mean order parameter of 0.92 and standard deviation of 0.09 was reported in solution, which is in remarkably good agreement with our solid-state analysis. However, the reported timescales of motion are different, but they are not comparable because they correspond to physically distinct models.

The range in which our GAF timescales fall is tens to hundreds of nanoseconds, similar to those obtained in a recent analysis of microcrystalline GB1 (33). The R_1ρ used in our solid-state study are highly sensitive to this timescale, whereas these timescales are essentially invisible to the methods applied in the solution-state study.

For example, consistently slower dynamics observed in the inner beta-strands here are not seen in the solution state. The solution-state analysis ascribes some fast motions on a low nanosecond timescale, but such motions would likely be incorporated into our GAF timescales. Here we clearly show that solid-state NMR relaxation can therefore provide dynamics information that is unavailable from solution data.

Conclusions

In conclusion, we have implemented a new approach that allows determination of structure and dynamics of a large paramagnetic metalloprotein using solid-state NMR. We have shown that deuteration, 100% amide reprotonation and ultrafast MAS allow the rapid and sensitive acquisition of an extensive set of ¹⁵N and ¹³C relaxation rates on SOD in its diamagnetic (Cu⁺) and paramagnetic (Cu²⁺) forms. From these data, PREs were obtained and employed as structural constraints for the determination of the protein structure. When added to ¹H-¹H distance restraints, they were shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion were obtained by a Gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecules, and interpreted in relation to backbone structure and metal binding.

The system we chose for this study is eminently suitable for the development of approaches for PRE determination and application in solid-state NMR on account of the ability to control the oxidation state of an intrinsically bound ion. Given that metalloproteins are common in biochemistry, and include many large (or membrane-bound) proteins that have metal-containing cofactors that can be manipulated into diamagnetic and paramagnetic forms, the ability to exploit quantitative PREs for structure refinement is potentially very useful (52). The method developed here is also directly compatible with the use of covalently attached paramagnetic tags (16, 19) in otherwise diamagnetic proteins. During the revision phase of this article, a paper has appeared showing that PREs induced in the solid-state by attaching a paramagnetic chelator to multiple cysteine mutants can be used in addition to TALOS restraints to determine the fold of the model protein GB1 (53). Moreover, the detail with which we have been able to characterize the internal dynamics is a clear indication that solid-state NMR relaxation is a powerful technique which we expect to make an impact in the study of systems not amenable to solution-state analysis. There exist many membrane-bound and fibrillar systems of medical or pharmaceutical interest in which such information may be very useful.

Methods

Sample Preparation.

The U-[²H,¹³C,¹⁵N] SOD samples were prepared essentially as previously described (13), but also metallated with Cu²⁺. In order to obtain a sample of Cu⁺,Zn²⁺-SOD, the Cu²⁺ form was reduced by the addition of 1.5 molar equivalents of iso-ascorbic acid under nitrogen atmosphere to avoid subsequent oxidation. The reduction was carried out prior to crystallization (also under nitrogen atmosphere) as in previous work.

NMR Spectroscopy.

All relaxation measurements were performed using a 19.9 T (¹H Larmor frequency 850 MHz) wide-bore Bruker Avance III spectrometer, and all experiments used 60 kHz MAS, with a sample temperature of 13 °C. The pulse sequences used for measurement of relaxation rates are shown in Fig. S1. The experimental PREs were obtained by subtracting the diamagnetic R₁ from the paramagnetic R₁. The (H)NHH_RFDR spectrum (τ_mix = 3.3 ms) was recorded for Cu⁺,Zn²⁺-SOD only, and was automatically assigned according to the procedure described in (13). PRE-derived distance restraints were then applied, and a final structure calculation performed using the (H)NHH_RFDR assignments as generated above.

For the fitting of dynamics models to relaxation data, for each site ¹⁵N R₁ and ¹⁵N R_1ρ were calculated according to well-known formulae for dipolar and CSA relaxation, reproduced in the SI Text (30, 33).

A full description of sample preparation, NMR spectroscopy, data analysis and structure calculation is provided as SI Text.