Xingyu
Lu
ab,
Huilan
Zhang
ab,
Manman
Lu
ab,
Alexander J.
Vega
a,
Guangjin
Hou
*ab and
Tatyana
Polenova
*ab
aDepartment of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA. E-mail: luxingyu@udel.edu; zhang@udel.edu; lumm@udel.edu; lexvega@comcast.net; hou@udel.edu; tpolenov@udel.edu; Fax: +1-302-831-6335; Tel: +1-302-831-1968
bPittsburgh Center for HIV Protein Interactions, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA
First published on 7th January 2016
Experimental characterization of one-bond heteronuclear dipolar couplings is essential for structural and dynamics characterization of molecules by solid-state NMR. Accurate measurement of heteronuclear dipolar tensor parameters in magic-angle spinning NMR requires that the recoupling sequences efficiently reintroduce the desired heteronuclear dipolar coupling term, fully suppress other interactions (such as chemical shift anisotropy and homonuclear dipolar couplings), and be insensitive to experimental imperfections, such as radio frequency (rf) field mismatch. In this study, we demonstrate that the introduction of window delays into the basic elements of a phase-alternating R-symmetry (PARS) sequence results in a greatly improved protocol, termed windowed PARS (wPARS), which yields clean dipolar lineshapes that are unaffected by other spin interactions and are largely insensitive to experimental imperfections. Higher dipolar scaling factors can be attained in this technique with respect to PARS, which is particularly useful for the measurement of relatively small dipolar couplings. The advantages of wPARS are verified experimentally on model molecules N-acetyl-valine (NAV) and a tripeptide Met-Leu-Phe (MLF). The incorporation of wPARS into 3D heteronuclear or homonuclear correlation experiments permits accurate site-specific determination of dipolar tensors in proteins, as demonstrated on dynein light chain 8 (LC8). Through 3D wPARS recoupling based spectroscopy we have determined both backbone and side chain dipolar tensors in LC8 in a residue-resolved manner. We discuss these in the context of conformational dynamics of LC8. We have addressed the effect of paramagnetic relaxant Cu(II)-EDTA doping on the dipolar coupling parameters in LC8 and observed no significant differences with respect to the neat sample permitting fast data collection. Our results indicate that wPARS is advantageous with respect to the windowless version of the sequence and is applicable to a broad range of systems including but not limited to biomolecules.
One approach to overcome the inaccuracies in the heteronuclear dipolar coupling measurements is the phase-alternating R-type symmetry (PARS) method reported by us recently.16 In this technique, the phase alternation introduced to the R-symmetry elements results in a drastic reduction of the residual 1H CSA term, making PARS particularly beneficial at high magnetic fields (18.1 T and higher). For fast MAS conditions (60 kHz and higher), a cross-polarization with variable contact (CPVC) experiment was demonstrated to yield accurate dipolar couplings.31 CPVC was shown to be insensitive to the rf field inhomogeneity.
The motivation for the current study was to establish a heteronuclear dipolar recoupling method that would not only provide efficient suppression of the 1H CSA but also lack sensitivity to the 1H rf field imperfections, and be applicable over a wide range of MAS frequencies. As detailed below, the introduction of windowed delays into the appropriate-symmetry PARS sequences produces a method that exhibits the desired behavior. We call this approach windowed-PARS, or wPARS for short.
The implementation of wPARS recoupling as a non-constant time 2D sequence is illustrated in Fig. 1a. The recoupled 1H–13C/15N dipolar evolution is modulated by the T2′ decay of the 13C/15N spins during the t1 period, which causes additional line broadening in the 1H–13C/15N wPARS dipolar spectrum. Usually the decay rate of the T2′ relaxation is much slower than the magnitude of the 1H–13C/15N dipolar couplings expressed in frequency units (ca. 10–20 kHz). Therefore, in practice the T2′ decay of 13C/15N spins does not affect the accuracy of the wPARS dipolar measurements and is simply represented by an empirical line broadening parameter used during the fitting of the wPARS dipolar line shape. Within this regular non-constant time wPARS scheme, the R-symmetry-based block, RN0, and the π-phase shifted R-symmetry block, RNπ, are applied on the 1H channel in an alternating fashion. The phases of the π pulses, which comprise the standard RNvn cycle RN0, alternate between ϕ and −ϕ, where ϕ = πν/N, and RNπ has its phases shifted to ϕ + π and −ϕ + π. Both RN0 and RNπ efficiently reintroduce the 1H–13C/15N dipolar interactions as well as 1H CSA (but with an opposite sign), since they possess the same symmetry properties, i.e. l = 2 and λ = 1. In the meantime, π pulses are applied on the X channel at the end of each RN block, and the inverse operation on the 13C/15N spins can restore the refocused 1H–13C/15N dipolar interactions but not the 1H CSA interaction. The windowless PARS technique is thus anticipated to yield accurate dipolar couplings whereas 1H CSA interaction is fully suppressed. To improve the PARS sequence, a finite window (τwin) with the fraction of fwin (0 ≤ fwin < 1) is inserted in the middle of the basic RN element (τ) splitting it into two π/2 pulses. This has the following consequences:
(1) higher rf field is required to obey the symmetry rules that ensure selection of the desired interactions, and the 1H rf field imperfection effect is then partly suppressed;
(2) the effective dipolar scaling factors of the basic RN sequences are improved by splitting the π pulses into two π/2 pulses, because the scaling factor is proportional to the integral of the reduced Wigner element whose maximum is π/2 for the single-quantum heteronuclear dipolar terms {l, m, λ, μ} = {2, ±2, 1, ±1}.7 Therefore, the scaling factor may be improved by turning the rf field off near π/2 pulses to give the single-quantum operator more time to accumulate in this favorable region. We note that the larger scaling factors in a recoupling sequence are useful when measuring relatively small dipolar couplings.
Fig. 1d shows the calculated scaling factors of the single-quantum R-symmetry heteronuclear dipolar recoupling sequences as a function of the window fraction of the RN element. The scaling factor at fwin = 0 corresponds to the regular windowless PARS or RN-DIPSHIT sequences. It can be seen that the scaling factors increase linearly with the window fraction, and, for instance, 26.6% enhancement can be achieved for R1013 with fwin = 0.5. The dependence of scaling factors on the required 1H recoupling rf field strength for various window fractions is shown in Fig. 1e. The approximately exponential function suggests that efficient enhancement of the scaling factors occurs when the strength of the rf field is less than four times that of the conventional windowless PARS sequence, which corresponds to the window fraction of 0.75.
For application to large or complex systems, an additional isotropic chemical shift dimension is required to attain the necessary site resolution, and the 3D implementations of wPARS-based dipolar recoupling are illustrated in Fig. 1b and c, where heteronuclear 15N–13C and homonuclear 13C–13C magnetization transfers are introduced through SPECIFIC-CP and fpRFDR, respectively.
The numerical simulations indicate that the replacement of RN elements by windowed pulses can reduce the sensitivity to 1H rf field imperfection. For this purpose, dipolar modulation curves were simulated for several 1H rf field strengths ranging from 80% to 120% of the nominal theoretical field strength required for a proper synchronization of the symmetry-based sequence. As shown in Fig. 2a, the stability of the recoupling sequence increases when the window fraction is increased. With the window fraction of 75% or 50%, the error in the dipolar coupling determined by the windowed PARS is less than 10%, even with a huge mismatch of 15% with respect to the theoretical rf field strength. Such tolerance for rf field mismatch is unusual in practical NMR experiments. A drawback is that the wPARS sequence with 75% window fraction requires the rf field strength to be 4 times higher than that used in the regular PARS sequence, e.g., rf strength of 200 kHz would be needed for 75% windowed-R1013 sequences at the MAS frequency of 10 kHz. Such fields are generally too high to be attained with most commercial MAS NMR probes, and hence 50% windowed RN sequences present a practically suitable compromise in terms of performance and implementation with the available hardware.
The comparison of the wPARS performance to that of other recoupling methods was analyzed by simulations using the same spin system, and the results are presented in Fig. 2b–d. As shown in Fig. 2b, wPARS, T-MREV8 and REDOR sequences can efficiently suppress the effect of 1H rf field imperfection. All three sequences employ a similar type of windowed recoupling elements, rendering them largely insensitive to 1H rf field imperfections. On the other hand, as shown in Fig. 2c, only PARS and wPARS suppress the influence from 1H CSA while other sequences are strongly dependent on the size and the orientation of the CSA tensor of the bonded proton. As discussed previously,14 with regular recoupling pulses on 1H, either windowed or windowless, the recoupled 1H CSA and 1H–15N dipolar interactions would interfere with each other during the rotor period, which results in distorted dipolar line shapes and associated errors in the corresponding H–X dipolar parameters. As shown in Fig. 2d, REDOR is in addition susceptible to the effects of 1H–1H homonuclear dipolar couplings. This sensitivity stems from the ability of REDOR to partially recouple the homonuclear dipolar term even in the hard pulse limit, which is why the application of REDOR is limited to the isolated spin-pair cases or extensively deuterated systems.55 On the contrary, for R-symmetry based recoupling sequences, such as R1013, the restricted selection rules result in efficient averaging of the 1st-order homonuclear dipolar couplings while the heteronuclear dipolar terms are reintroduced. It is also worth noting that wPARS exhibits almost no dependence on 1H–1H homonuclear dipolar couplings up to 30 kHz, while PARS or T-MREV8 shows slight dependence, which suggests that more efficient suppression on higher order 1H–1H homonuclear dipolar couplings can be obtained by wPARS.
Generally, among the dipolar recoupling methods discussed above, the windowed PARS sequence exhibits the most favorable properties, including efficient suppression of 1H–1H homonuclear dipolar and 1H CSA interactions, as well as reduced sensitivity to rf field inhomogeneity. wPARS sequences do not require fast MAS conditions (MAS frequencies greater than 60 kHz), in contrast to CPVC based techniques. These features arguably make wPARS an optimal method for accurate measurements of heteronuclear couplings in a wide range of systems, including large biomolecules, as demonstrated below.
Fig. 3a and c shows the plots of errors in the 1H–15N dipolar couplings of NAV and MLF determined experimentally from wPARS spectra recorded with different window fractions, as a function of the 1H rf field mismatch. The results demonstrate that, in agreement with numerical predictions, wPARS recoupling efficiently suppresses the influences from 1H rf field imperfection and gives rise to undistorted 1H–15N dipolar powder patterns. The experimental error depends on the window fraction to the degree predicted by the numerical simulations. In agreement with theory, the larger the window inserted into the RN elements, the lower the sensitivity of the wPARS to 1H rf field imperfection. Typically, a 50% window fraction in PARS resulted in errors of less than 5% in the 1H–15N dipolar couplings, when the mismatch of the rf field strength was in the range of ±10% from the theoretical value. It is important to note that the 5% error is generally of the same order as the systematic measurement errors. Therefore, 50% windowed PARS is basically insensitive to common 1H rf field imperfections. A careful examination of the dipolar lineshapes recorded in the 50% wPARS with the matched and mismatched (by −15%) 1H rf field indicates that the effect of the 1H rf field imperfection is essentially negligible, except for a slight change in the central peak intensity (see Fig. 3b and d). The latter is the result of the contributions from partially unrefocused higher-order 1H CSA Hamiltonian terms. As expected from numerical simulations, the introduction of the 50% window resulted in an increase of the heteronuclear dipolar scaling factors by ca. 25% compared to the conventional PARS spectra. As noted earlier, this improvement is particularly important for the measurements of small 1H–13C/15N dipolar couplings (across multiple bonds or partially averaged due to dynamics).
We note that an R element can be replaced with windowed pulses in the context of the conventional RN-DIPSHIFT experiment when 1H CSA is negligible in the system of interest. Not surprisingly, a windowed RN-DIPSHIFT sequence also works well for the suppression of 1H rf field imperfections and also exhibits increased dipolar scaling factors (see Fig. S1, ESI†).
It is of interest to measure both 1H–15N and 1H–13C dipolar parameters as the latter report on the dynamics of side chains in proteins. 1H–13C dipolar parameters can be conveniently measured in a 3D experiment, where the dipolar recoupling period is followed by a 13C–13C mixing step, such as fpRFDR, as reported previously32 and shown in Fig. 1c in the context of wPARS recoupling sequences.
To verify that performance of wPARS in the context of the 3D wPARS-fpRFDR experiment, we have compared 3D and 2D R1013-based 1H–13C wPARS spectra on the MLF sample (Fig. 1a). As shown in Fig. S2 (ESI)†, the 1H–13Cα dipolar line shapes for all residues in MLF extracted from the 2D spectrum are fully consistent with those obtained from 13Cα–13Cα, 13Cα–13Cβ and 13Cα–13Co cross-peaks in the 3D experiment. This result indicates that the fpRFDR mixing does not introduce any artifacts into the spectra, in accordance with the previous findings for a 3D CPVC-RFDR experiment.32
To experimentally examine the application of wPARS in proteins, we have performed 3D experiments on dynein light chain, LC8, which has been extensively characterized in our laboratory.24,50 We have examined two samples, nanocrystalline U–13C,15N-LC8 and the same preparation but doped with 5 mM Cu(II)–EDTA. The latter sample was studied by us previously in the context of experiments combining nonuniform sampling and paramagnetically assisted condensed data collection (NUS-PACC).51 The introduction of paramagnetic dopants permits large time savings due to dramatic reductions in T1, which are particularly well realized in the context of fast MAS, where low-power decoupling can be used.47–49 We have reported previously that the presence of 5 mM Cu(II)–EDTA does not have a significant effect on the LC8 spectra.51
Fig. 4 shows expansions around several regions of the first 2D 13C–13C plane extracted from the 3D wPARS spectrum of LC8 prepared without Cu(II)–EDTA. The corresponding 1H–13Cα dipolar line shapes for each residue were extracted along the third dimension in both 3D spectra. We obtained a total of 62 well-resolved wPARS dipolar patterns in each of the two samples, including 50 backbone and 12 side chain 1H–13C line shapes. Representative experimental and fitted 1H–13Cα dipolar line shapes for three LC8 residues are plotted in Fig. 4c. Note that the errors are less than ±5%.
Fig. 5a shows the comparison of backbone 1H–13C dipolar order parameters (DOP or S) of U–13C,15N-LC8 prepared with and without Cu(II)–EDTA, plotted as a function of the residue number. The rigid-limit dipolar coupling for one-bond 1H–13C with a bond length of ∼1.09 Å is 22.8 kHz. Molecular motions occurring on timescales of 10−9–10−6 s give rise to reduced dipolar couplings. The results indicate that most 1H–13C S are larger than 0.85, including those for the loop regions. Only a few residues, A21, E30 and K87, have DOP less than 0.85, indicating that these are somewhat flexible at the experimental temperature of −18 °C. The S values derived from wPARS are in overall strong agreement with our prior report, where S were derived from the conventional DIPSHIFT experiment.15 Comparing the results for the LC8 samples prepared with and without Cu(II)–EDTA, we find that, for the majority of the residues, Cu2+ had no noticeable effect on the dipolar order parameters, with the deviations being generally smaller than 0.06. These deviations correspond to 1.5 kHz for dipolar coupling, which is of the order of the fitting errors. The 1H–13C dipolar line shapes for 50 representative backbone residues in LC8 samples with and without Cu doping are shown in Fig. S3 (ESI†).
There are 9 residues whose peaks are resolved and that show deviations in 1H–13C dipolar order parameters greater than 0.06 in the presence of Cu(II)–EDTA, as illustrated in Fig. 5a. These residues are displayed in the context of the LC8 3D structure in Fig. 5b. The dipolar couplings measured for these residues in the Cu(II)–EDTA doped sample are greater than those without copper. Of these, E30, E45, and Q80 are located on the surface of LC8, and their side chains may be interacting with the dopant, affecting the dipolar couplings. Another possible source of error for these residues is the faster decay of 13C T2 relaxation during the RFDR period caused by the presence of paramagnetic ions (see Fig. 1c). This is illustrated also in Fig. S4 (ESI†), showing the corresponding dipolar line shapes. There are no correlations between the relatively large dipolar order parameter deviations for the 9 residues and their chemical shift differences (Fig. S5, ESI†). The chemical shift perturbations are generally smaller than 0.2 ppm, in agreement with our previous observations.51
The combination of wPARS with fpRFDR allows us to determine not only the backbone dipolar order parameters but also those for the aromatic side chains, in a single 3D experiment. The aromatic side chain dipolar tensors are a sensitive probe of dynamics, and in LC8 many of the aromatic residues are mobile.32 The analysis of the side chain dynamics of the aromatic residues in U–13C,15N-LC8 is shown in Table S2 and Fig. S6 (ESI†). As discussed in our recent report,32 in the analysis of dipolar interactions of aromatic side chains, the dipolar asymmetry parameters ηD cannot be ignored. In Table S2 (ESI†), the 1H–13C dipolar couplings and their corresponding dipolar asymmetry parameters ηD are shown for different residues. For H55 and W54 residues, the average 1H–13C dipolar couplings are around 21 kHz, close to the rigid-limit 1H–13C coupling of 22.8 kHz, and the corresponding ηD values are generally smaller than 0.4. This observation is consistent with our previous findings that these residues are nearly static.32 On the other hand, for F76, Y41 and Y75, the 1H–13C dipolar couplings are smaller than 15 kHz, and the corresponding asymmetry parameters are 0.6 or higher. These values indicate that there are π-jumps occurring around the Cβ–Cγ (x-) axis of the aromatic ring, also consistent with our previous report.32 In this study,32 at room temperature, one additional kind of side chain motion for Phe and Tyr residues was also discussed, a ring displacement by a small angle along the z-axis. Such motions were not detected in our present study likely because the wPARS experiments were conducted at −18 °C, where the motions are more restricted compared with room temperature conditions. With respect to the influence of paramagnetic Cu(II)–EDTA doping on dipolar interaction parameters for the side chains of LC8, there are no significant differences observed in the presence of Cu(II)–EDTA, as shown in Table S2. This is in line with our findings for the 1H–13C backbone order parameters.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5cp07818k |
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