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High resolution 13C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all 15N, 13C′, 13Cα and 13Cβ sites are resolved in 13C–13C and 15N–13C spectra, with significant improvement in T2 relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T2 values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that 13Cα T2′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.
Deuteration is a common strategy employed in protein solution NMR to improve the spectral resolution and sensitivity by increasing the transverse relaxation times (T2) and suppressing the 1H–1H scalar couplings (Gardner and Kay 1998). The combination of triple resonance experiments with 2H, 13C, 15N labeled samples is essential for the solution structure determination of large proteins, but the substitution of deuterons for protons depletes the number of protons available for distance restraints. As a result, several deuterium-labeling schemes that produce molecules with different patterns of incorporation have been employed (Gardner and Kay 1998).
Initial applications of deuteration to solid-state NMR (SSNMR) have been reported in the literature, including both the study of protein dynamics and 1H–1H distance measurements, based on the detection of the deuterium and proton nucleus, respectively (Hologne et al. 2006; Linser et al. 2008). The relaxation and lines shape of the deuterium quadrupole, which are sensitive to the local dynamics, have also been used to obtain information on different motional behaviors (Hologne et al. 2006). The large gyromagnetic ratio of the 1H nucleus provides high sensitivity to measure 1H–1H distances that are very useful for structure calculations, where deuteration has been used to eliminate the strong 1H homonuclear dipolar couplings (Paulson et al. 2003). Recently, NMR samples with paramagnetic ion doping under fast MAS have shown enhanced sensitivity with fast data acquisition (Wickramasinghe et al. 2009). A set of low power 1H decouplings also showed high resolution spectra of several proteins under fast MAS (Kotecha et al. 2007; Vijayan et al. 2009). Proton dilution from deuteration and back-exchange, combined with fast MAS and low power 1H decoupling, made it possible to obtain enough 1H–1H distance constraints to calculate a full protein structure (Zhou and Rienstra 2008).
The application of deuterated samples for 13C detection in typical solid-state NMR experiments, such as 13C–13C or 15N–13C correlations, has been investigated in the case of membrane proteins such as bacteriorhodopsin (Varga et al. 2007). Although some difficulties might be expected for 1H–13C cross-polarization (CP) with deuterated samples due to the dilution of the 1H bath, Morcombe et al. demonstrated that CP efficiency is still reasonably efficient, the effect on the 1H and 13C T1 relaxation times is surprisingly little, and no statistically significant broadening of the 13C line widths was observed in the case of ubiquitin (Morcombe et al. 2005).
Here we present the effect of deuteration on chemical shifts, T2 relaxations times, spin diffusion rates, and 1H decoupling requirements for a deuterated protein, the beta-1 immunoglobulin binding domain of protein G (GB1), in high resolution 13C-detected solid-state NMR spectra. Our results show significant improvement in T2 relaxation times even with low proton-decoupling power that will make it possible to perform J-based SSNMR experiments (Chen et al. 2006, 2007) to improve resolution of non-crystalline samples. The improvement in the T2 relaxation times and resolution will facilitate the sequential backbone assignments of large systems in which peak separation represents a big challenge, and exchangeable protons or water protons will enhance the selective detection of polar residues or residues that are close to water molecules. Therefore, the application of deuteration to SSNMR shows great potential in the structure determination of large protein systems that are of high interest and also in the evaluation of water accessibility on protein complexes and aggregates.
1H, 13C, 15N-labeled GB1 (U-CN-GB1) and 2H, 13C, 15N-labeled GB1 (U-CDN-GB1) were prepared according to previously published procedures (Franks et al. 2005); (Zhou et al. 2007). The deuterated protein was purified in H2O, so the exchangeable deuterons were replaced by protons. Nanocrystalline samples were precipitated with 2-methyl-pentane-2,4-diol (MPD) and isopropanol (IPA; Franks et al. 2005). A total of 18 mg material with 6 mg (1.0 μmol) U-CDN-GB1 was packed into a standard wall 3.2 mm MAS rotor with rubber disks for maintaining hydration. About 5 mg (0.9 μmol) U-CDN-GB1 and 4 mg solvents were packed into a 1.6 mm MAS rotor, and around 5 mg (0.9 μmol) U-CN-GB1 and 4 mg solvents were packed into another 1.6 mm MAS rotor.
NMR experiments were carried out on a 750 MHz Varian INOVA spectrometer with a 3.2 mm BioMAS probe (Stringer et al. 2005) and a 1.6 mm BioFastMAS probe (Varian, Inc.). For the BioMAS probe, the π/2 pulse widths for 1H, 13C, 15N were 2.7, 4.5, 7.0 μs, respectively. For the BioFastMAS probe, the π/2 pulse widths for 1H, 13C were 1.7, 2.8 μs, respectively. The MAS rates were 12.5 kHz on the BioMAS probe and 18–40 kHz on the BioFastMAS probe. For 2D 13C–13C and 15N–13C experiments, the dipolar assisted rotational resonance (DARR) scheme (Takegoshi et al. 2001) was used for 13C–13C mixing. For 2D NcaCX experiments, a selective SPECIFIC CP (Baldus et al. 1998) was used for polarization transfer from 15N to 13C. Typical 1H decoupling at 70 kHz was used for both t1 and t2 evolutions in 2D experiments at 12.5 kHz MAS. For 2D J-MAS CACO IPAP experiments (based on the CTUC COSY experiment Chen et al. 2006, 2007) acquired at 32 kHz MAS, 110 kHz SPINAL 64 (Fung et al. 2000) decoupling was used during the constant-time intervals and acquisition. Average echo T2s (typically referred to as T2′ (Lesage et al. 1999)) values for C′ and Cα at different decoupling conditions were measured using a 1D CP Hahn-echo (Hahn 1950) experiment with a 180° soft pulse on C′ or Cα resonance to remove the effect of 13C–13C J-couplings (Li et al. 2006; the pulse sequence is shown in Fig. 1). It has been reported that the linewidths of deuterated methyl groups can be improved by 2H decoupling (Agarwal et al. 2006), so we measured T2 values of C′, Cα and methyl groups of U-CDN-GB1 with low power 2H WALTZ-16 (Shaka et al. 1983) decoupling (~1.2 kHz) with a 1.6 mm 1H–13C–15N–2H magic-angle spinning probe on 500 MHz (1H frequency) at 32 kHz MAS and found significant T2 improvement in methyl groups as expected. Chemical shifts were referenced externally with adamantane with the downfield 13C resonance of 40.48 ppm on the DSS scale (Morcombe and Zilm 2003).
2D spectra were processed with NMRPipe (Delaglio et al. 1995) and were analyzed with Sparky program (T. D. Goddard and D. G. Kneller, University of San Francisco). Back linear prediction and polynomial baseline correction were applied to the direct dimension. Zero filling and Lorentzian-to-Gaussian apodization were used for each dimensions before Fourier transformation. Detailed acquisition and processing parameters are included in figure captions.
To evaluate the effect of deuteration on 13C relaxation properties, 13C CP Hahn-echo experiments were carried out for deuterated and protonated GB1 under different spinning rates and different 1H decoupling sequences with various radio-frequency (RF) field strengths. The field strengths were selected to avoid rotary resonance conditions at high MAS rates by picking the local maximums of signal intensities while arraying the CW decoupling strengths (Ernst et al. 2003). Table 1 summarizes the 13C T2′ values of U-CDN-GB1 and U-CN-GB1. At the moderate spinning rates (12.5 and 18 kHz), the 13Cα T2′ of U-CDN-GB1 is almost twice the U-CN-GB1 with optimized SPINAL decoupling (26 vs. 13 ms). Even at the high spinning rate of 40 kHz, both the 13Cα and 13C′ T2′ of U-CDN-GB1 are much longer than for U-CN-GB1. Therefore, the removal of 13C–1H dipolar couplings by deuteration indeed improves the 13C T2′ significantly. In terms of J-coupling effect from 2H, we compared T2 values of C′, Cα and methyl groups of U-CDN-GB1 under low power 2H WALTZ-16 decoupling (~1.2 kHz) at 32 kHz MAS, and found no improvement in C′ (50 ms) and Cα (25 ms), but great enhancement in methyl groups from 21 to 50 ms. In addition to the echo T2 values, the directly observed 13C linewidths are greatly narrowed. Figure 2 and and33 show well-resolved 13C–13C 2D and NcaCX 2D spectra of U-CDN-GB1. Typical 13C linewidths in the 2D spectra are 0.2–0.4 ppm, to which 13C–13C J-couplings, present in uniformly labeled samples, contribute 35–55 Hz (0.2–0.3 ppm). Especially apparent in the carbonyl region of the NcaCX 2D spectra (Fig. 3b) where 13C′ linewidths are less than 0.2 ppm, so that some N–C′ crosspeaks actually show the splitting of ~50 Hz corresponding to JCOCA (such as Y3, K4, D26, V39, F52, T55).
The assignments of U-CDN-GB1 were completed by comparing the 2D spectra with the ones of U-CN-GB1, using the published chemical shift of U-CN-GB1 and assuming that the upfield shift of 13C chemical shift caused by deuterium is around 0.5–1.0 ppm. The 13C′, 13Cα, 13Cβ, 15N of all the residues have been assigned. As shown in the 13C–13C 2D spectrum with short 50 ms mixing time (Fig. 2a), there are clearly resolved Ala Cα-Cβ, Gly Cα-C′, Thr Cα-Cβ and Cβ-Cγ2 crosspeaks. An expansion of the Cα-Cβ crosspeaks of all other residues is shown in Fig. 2b. The residues that are in β-strand have stronger Cα-Cβ peaks than those in helix, and the peaks from polar and aromatic residues have higher intensities than those from nonpolar residues. At long 300 ms mixing time (Fig. 2c, d), the spectrum showed more correlations between the sidechains and backbone carbons of polar residues, and also some inter-residual correlations in which at least one residue is polar or aromatic. Similar crosspeak patterns occurred in NcaCX 2D spectra (Fig. 3). Almost all the N–C′, N–Cα, N–Cβ peaks are well resolved in the spectrum at short mixing time, with some N–Cγ (Asn, Asp), N–Cδ (Gln, Glu) and N–Cε (Lys) peaks for the residues with exchangeable-proton sidechains. At the long mixing time, inter-residue N–C peaks show up and mainly involved with polar residues. In the expanded region of NcaCX 2D spectrum with 300 ms mixing (Fig. 3d), crosspeaks between Leu and other residues are clearly weaker than those between polar residues. Furthermore, some cross-peaks appear in the region of 110–140 ppm of 13C chemical shift, corresponding to the correlations between amide nitrogens and sidechain carbons of Y3, Y33, Y45 and W43, which also have exchangeable protons on the side chains. Overall, most sidechain 13C of polar residues and some of the nonpolar and aromatic residues were assigned, and the deviations of these assignments were within 0.1 ppm. Therefore, the deuterated protein samples not only maintain the capability of assigning all the backbone resonances that are important for determining the secondary structures, but also strengthen the selection of sidechains of polar residues that are closely related to protein functions or interactions between proteins. Particularly in solid-state NMR, this feature would be valuable for assigning loop regions or channel lining interfaces of membrane proteins, since those regions are usually abundant of polar residues.
To confirm our assumptions and characterize in more detail the upfield isotope shifts from deuterium, we compared the chemical shifts of U-CDN-GB1 and U-CN-GB1, as summarized in Table 2. Δδ(D)calc are calculated by the equation (Venters et al. 1996):
where nΔδ(D) represents the n-bond isotope effect per deuteron and dnb is the number of deuterons n bonds away from the detected nucleus. The average deuterium effects are ~0.1 ppm for 13C′, ~0.3 ppm for 13Cα and ~0.9 ppm for 13Cβ. Generally, the deuterium effects in solid-state NMR are slightly smaller (0.1–0.2 ppm) than the values in solution NMR. For each site, the deuterium effects are mostly consistent (deviations within 0.1 ppm), except a few outliers with deviations larger than 0.4 ppm (Thr, Trp, Tyr 13Cβ, Lys 13Cδ and 13Cε). Such deviations were also observed in solution NMR (Venters et al. 1996), which could result from subtle environmental changes around these sites that are next to either hydroxyl groups, amine groups or aromatic rings.
While all multidimensional experiments gain from the increase in signal power associated with longer T2 values, scalar-coupling-driven correlation experiments will additionally benefit from the slower decoherence rates during the relatively long echo periods necessary for coherence transfer through the J coupling. Indeed, during the last decade a growing number of 2D and 3D correlation experiments have emerged that make use of the indirect spin–spin coupling to map out through-bond connectivity in solid-state proteins (Chen et al. 2007; Chen et al. 2006; De Paepe et al. 2003; Detken et al. 2001; Linser et al. 2008). The vast majority of these experiments have been implemented under conditions of high-power proton decoupling (>150 kHz), where the value of the echo T2′ is maximized (De Paepe et al. 2003). Deuteration will allow the use of lower power decouplings while maintaining longer T2′ values (Agarwal et al. 2006), as we exploit in the implementation of the J-MAS CACO IPAP correlation experiment (Chen et al. 2006, 2007) shown in Fig. 4. This J-based correlation spectrum was acquired at 32 kHz MAS with only moderate (110 kHz, 75% of the full power—150 kHz) proton decoupling. The combination of the constant-time evolution in the indirect dimension (Chen et al. 2006, 2007) and the in-phase anti-phase selection in the direct dimension (Bermel et al. 2005) leads to a highly resolved 2D spectrum and deuteration paired with low power proton decoupling that is clearly compatible even with challenging J-based transfer experiments.
One significant practical aspect of these studies is that the 1H–13C CP conditions for the U-CDN-GB1 sample are much more sensitive than those for U-CN-GB1. This is the case even with a large tangent amplitude ramp applied, and thus the CP amplitudes need careful optimization under the condition where 2D experiments would be performed. Moreover, the overall instrumental stability is critical, in addition to the optimization of 1H decoupling to achieve optimal T2′ values. Figure 5 shows that U-CDN-GB1 Cα signal intensities had less variation with different pulse widths and phase angles of SPINAL 64 decoupling that those of TPPM decoupling. Therefore, SPINAL decoupling is much less sensitive to pulse width and phase angle than TPPM decoupling. We also found that SPINAL decoupling is less sensitive to 1H carrier frequency than TPPM decoupling (data not shown). Figure 6 compares the NcaCX 2D spectra of U-CDN-GB1 at 12.5 kHz MAS with SPINAL and TPPM decouplings. The 15N linewidths are significantly better using SPINAL decoupling than using TPPM decoupling (0.5 vs. 1.2 ppm for D40 15N). Therefore, SPINAL decoupling is a better choice for deuterated protein samples. In addition, low-power XiX decoupling provides reasonable T2′ values at fast MAS (Table 1), and thus it is also a viable choice in fast spinning regime.
We have demonstrated 13C-detected solid-state NMR spectroscopy of deuterated GB1. Deuteration provides a significant improvement in 13Cα T2′ by eliminating the 13C–1H dipolar couplings. T2′ enhancement makes experiments that require long T2′ values feasible, such as J-based experiments, especially for large proteins in higher magnetic field. Unique crosspeak patterns that result from exchangeable protons on sidechains of polar residues reduce the overlap in the methylene and methyl regions and provides easier identification of polar residues in large proteins, which usually play important roles in protein functions and/or in the interactions with other proteins.
The authors thank the National Institute of Health (NIGMS GM075937 and GM073770, S10RR023677) and the National Science Foundation (CHE-0848607) for funding, the Caja Madrid Foundation and Agusti Pedro Pons graduate fellowships to GC, and John J. Shea and John M. Boettcher for the sample preparation.
Ming Tang, Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Gemma Comellas, Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
Leonard J. Mueller, Department of Chemistry, University of California, Riverside, CA 92521, USA.
Chad M. Rienstra, Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA. Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA. Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.