Fractional deuteration applied to biomolecular solid-state NMR spectroscopy
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Solid-state Nuclear Magnetic Resonance can provide detailed insight into structural and dynamical aspects of complex biomolecules. With increasing molecular size, advanced approaches for spectral simplification and the detection of medium to long-range contacts become of critical relevance. We have analyzed the protonation pattern of a membrane-embedded ion channel that was obtained from bacterial expression using protonated precursors and D2O medium. We find an overall reduction of 50% in protein protonation. High levels of deuteration at Hα and Hβ positions reduce spectral congestion in (1H,13C,15N) correlation experiments and generate a transfer profile in longitudinal mixing schemes that can be tuned to specific resonance frequencies. At the same time, residual protons are predominantly found at amino-acid side-chain positions enhancing the prospects for obtaining side-chain resonance assignments and for detecting medium to long-range contacts. Fractional deuteration thus provides a powerful means to aid the structural analysis of complex biomolecules by solid-state NMR.
KeywordsAssignment Deuteration Ion channel MAS Solid-state NMR Structural constraints
Solid-state Nuclear Magnetic Resonance (ssNMR) combined with Magic Angle Spinning (MAS, (Andrew et al. 1958)) has in the last years made significant progress to study complex biomolecular systems including membrane proteins (Lange et al. 2006a; Ader et al. 2008; Bajaj et al. 2009; Ahuja et al. 2009; Shi et al. 2009; Etzkorn et al. 2007, 2010; Cady et al. 2010) or protein assemblies (Heise et al. 2005; Andronesi et al. 2008; Wasmer et al. 2008; Poyraz et al. 2010; Sun et al. 2009; Kumar et al. 2010; Jehle et al. 2010). In parallel, methods have been devised to determine entire three-dimensional structures from a single (Nomura et al. 1999; Rienstra et al. 2002; Lange et al. 2005; Manolikas et al. 2008; Bertini et al. 2010a) or a few (Castellani et al. 2002) protein preparations. With increasing molecular size, spectral resolution becomes critical for several aspects of the structure determination process. To deal with these challenges, multi-dimensional correlation experiments have been proposed and more elaborate isotope labeling schemes have been used (See Renault et al. 2010 for a recent overview). Some of the latter approaches simplify the spectral analysis to detect certain protein resonances but the essential process of structure determination, i.e., polarization transfer via C–C, C/N-HH-C (Lange et al. 2002)), or C/N–H-C (Seidel et al. 2005; Paepe et al. 2008; De Paepe et al. 2011)) spin moieties remains largely unaffected. At the same time, protein deuteration that has long been recognized as a powerful tool for macromolecular structural analysis by solution-state NMR (Englander et al. 1996; Gardner and Kay 1998) has been introduced in ssNMR for resolution enhancement of 1H solid-state NMR (Pines et al. 1976; McDermott et al. 1992; Zheng et al. 1993). In the last years, such approaches have been optimized to further reduce 1H line width (Chevelkov et al. 2006; Zhou et al. 2007; Linser et al. 2011), establish structural constraints (Reif et al. 2001; Paulson et al. 2003; Reif et al. 2003; Zhou et al. 2007; Huber et al. 2011; Varga et al. 2007) and to characterize protein-water interactions (Bockmann et al. 2005; Lesage et al. 2006). However, increasing levels of deuteration compromise the prospects to probe structurally relevant proton–proton distance constraints, affect relaxation times and may be prohibited by reduced protein expression levels in complex biomolecules such as membrane proteins.
In the following, we show that fractional deuteration (Rosen et al. 1996; Shekhtman et al. 2002; Otten et al. 2010) which can be readily obtained during protein expression by the combined use of protonated precursors and D2O provides a route to reduce spectral crowding and enhances the prospects to detect long-range correlations in standard ssNMR correlation experiments on complex biomolecules. For our study, we produced a fractionally deuterated version of a chimeric potassium channel (KcsA-Kv1.3) for which ssNMR resonance assignments have been reported (Schneider et al. 2008) and which has previously been characterized by ssNMR in different functional states in a lipid bilayer environment (Ader et al. 2008; Ader et al. 2009a, b). We analyzed the residual level of protein protonation and the effect of fractional deuteration on (13C,13C) as well as (15N,13C) and (1H,13C) ssNMR experiments. We show that a substantial reduction in proton density in [1H/2H,13C,15N] labeled KcsA-Kv1.3 influences the polarization transfer dynamics in the context of chemical-shift selective transfer. Because the residual protonation level favours peripheral amino-acid positions, establishing side-chain resonance assignments as well as the detection of long-range contacts, in particular between aromatic and methyl side-chains, is facilitated.
Materials and methods
Preparation of [1H/2H,13C,15N] KcsA-Kv1.3
Following the work of Legros et al. (Legros et al. 2000), the pQE32 expression construct (Lange et al. 2006a) was transformed into E.coli strain M15 prep4. For protein production, E.coli cells were grown on a medium containing protonated glucose and D2O. Cultures were adapted from initially 33–99% D2O over 3 days on small scale shaker flasks containing M9 minimal medium. The final culture was tenfold diluted into the expression culture. Protein expression was induced at 25°C by adding 0.5 mM IPTG at OD600 = 0.9. Cells were harvested as soon as the stationary phase was reached (5–6 h after induction). The protein was purified from 10 L of expression culture as described before (Lange et al. 2006a, b). Reconstitution into Asolectin liposomes was performed as described (Lange et al. 2006a, b), with minor changes. Briefly, the buffer of the purified channel protein was exchanged against 50 mM Sodium Phosphate, pH 7.4, 100 mM Sodium chloride, 4 mM n-decyl-ß-Dmaltopyranoside (DM) (Calbiochem) using a HiPrep 26/10 desalting column (GE Healthcare). Asolectin from soybean (Fluka) was resuspended in the same buffer and added at a 100/1 Asolection/KcsA-Kv1.3 molar ratio. This suspension was incubated for 2 h at room temperature. Subsequently, detergent was removed with Calbiosorb Adsorbent (Calbiochem). Liposomes containing [1H/2H,13C,15N]-labeled KcsA-Kv1.3 protein were pelleted by centrifugation at 134,000 × g for 2 h at 4°C.
All experiments were recorded using a 3.2 mm triple-resonance (1H,13C,15N) probe head at a static magnetic field of 16.4 T corresponding to 700 MHz proton resonance frequency (Bruker Biospin, Karlsruhe, Germany). 1H field strengths of 83.3 kHz were employed for 90° pulses and SPINAL64 (Ref. (Fung et al. 2000)) decoupling during evolution and detection periods. Initial (1H,13C) cross-polarization (CP) times were set to 80 μs to largely restrict polarization transfer to directly bonded 1H-13C spin pairs in (1H,13C) and (13C,13C) correlation experiments. For (15N,13C) correlation experiments, an initial (1H,15N) CP time of 800 μs was used. 15N-13C transfers were performed with a SPECIFIC-CP (Baldus et al. 1998) time of 3 ms, followed by homonuclear 13C-13C DARR (Takegoshi et al. 2001) mixing of 100 ms under 10.92 kHz MAS. (1H,13C) correlation experiments were conducted using frequency-switched homonuclear Lee-Goldburg (FSLG, Bielecki et al. 1989) decoupling at 83.3 kHz 1H field strength during the indirect 1H evolution period and the proton dimension was calibrated using regular (1H,13C) HETCOR spectra. For 1H-1H double-quantum (DQ) mixing, we employed the SPC5 recoupling scheme (Hohwy et al. 2002) with DQ excitation and reconversion times of 285 μs and a CP time as in the FSLG HETCOR experiments. To enhance rotational resonance recoupling for carboxyls and backbone Cα’s, MAS rates were set to 10.92 kHz or 8.5 kHz. Transfer among aromatic and aliphatic methyl side chains was enhanced using an MAS rate of 10 kHz. Mixing times ranged from 20 to 500 ms in (13C,13C) proton-driven spin-diffusion (PDSD) experiments. All experiments were performed with an effective sample temperatures ranging from +2°C to +7°C. Spectra were processed in Topspin (Bruker Biospin) and analyzed with Sparky (T. D. Goddard and D. G. Kneller, SPARKY 3, University of California, San Francisco).
Assignment and structural analysis
SsNMR resonance assignments for KcsA-Kv1.3 in lipid bilayers were taken from Ref. (Schneider et al. 2008). Since KcsA-Kv1.3 only differs by 11 turret residues from the 4 × 160 amino acid KcsA channel (Schneider et al. 2008) and in line with previous ssNMR work (Ader et al. 2008; Schneider et al. 2008; Ader et al. 2009b), the structure of the closed-conductive state of KcsA-Kv1.3 must share essential structural features with crystalline KcsA. Therefore, we created a structural homologue of the KcsA-Kv1.3 channel in the closed conductive state using the crystal structure of full length KcsA (PDB ID 3EFF, Uysal et al. 2009). Intra- and intermolecular 13C-13C correlations were then predicted using the KcsA-Kv1.3 model with an upper distance cutoff of 5 Å & 6 Å and, at the same time, taking into account the residual protonation pattern identified from ssNMR experiments. With these cutoff parameters (which were varied between 4 and 8Å) we observed the best overall agreement between experimental data sets and predicted cross peak patterns.
Identification of residual protonation pattern
The aliphatic region of the resulting spectrum is largely devoid of Cα-Cβ correlations (such as relating to Ile, Lys, Phe, Tyr or Asp residues), except for amino acids in which only one of the 13C positions is deuterated (Fig. 1a, red). For such protein residues (Ser, Thr, Cys, etc.) we observe, as expected for the short CP time (used in Fig. 1a), asymmetric correlation peaks. In line with earlier solution-state NMR studies (Rosen et al. 1996; Shekhtman et al. 2002; Otten et al. 2010), Cα positions of all amino acids are largely deuterated because the respective keto-acid undergoes a transamination reaction during synthesis (Nelson and Cox 2008). To further explore the residual 1H pattern, we conducted a conventional NCACB correlation experiment (Baldus 2002) using (13C,13C) DARR (Takegoshi et al. 2001) mixing (Fig. 1c). Similar to the results of the (13C,13C) correlation experiments, the NCA part of the spectrum largely agrees with data obtained on a protonated version (Supporting Figure 1) of the channel but the aliphatic region of the spectrum lacks several of the correlations that involve deuterated Cβ or Cγ positions. Indeed, Cβ positions of Glu, Gln, Pro and Arg that relate to α-Ketoglutarate as precursor in the biosynthetic pathway (Ref. (Nelson and Cox 2008), see also supporting table 1) are largely removed compared to the protonated case (Fig. 1c, red and Fig. 1b, green). Additional missing intensities relate to Cβ positions of Val and Ile, the aromatic amino acids of Phe, Tyr and His as well as the Cγ1 positions of Leu and Ile residues. On the other hand, pyruvate serves as a precursor to alkyl containing residues by direct incorporation (Ala, Val, Ile, Leu, Lys, etc.) or to aromatic amino acids and amino acids derived from Serine via other metabolites such as phosphoenol pyruvate and 3 phosphoglycerate (Supporting table 1). Hence, side chains of several amino acids containing alkyl groups are expected to exhibit sizable levels of protonation in line with our data. The protonation pattern at the remaining positions of amino acids is subject to residual protons from glucose itself and various intermediary steps that include cyclization, hydration, transamination or decarboxylation (Nelson and Cox 2008).
A more detailed analysis will be possible using topology schemes developed for solid-state NMR (Sakellariou et al. 2001) or using three-dimensional 1H-1H-13C or 1H-13C-13C experiments. A summary of the residual protonation pattern at the carbon sites identified from our CC/NCACX and DQ-SQ (1H,1H)-13C experiments is given in supporting table 1.
Assignments and structural constraints
Compared to the protonated case (Fig. 1b, green), fractional deuteration significantly reduces spectral complexity in complex biomolecules such the KcsA-Kv1.3 channel. We hence explored the use of such data for spectral assignment as well as for the structural analysis. Firstly, knowledge of the protonation pattern and the unique amino acid sequence of KcsA-Kv1.3 readily allowed us to obtain resonance assignments for Cys90 (Fig. 1a) not reported previously (Ader et al. 2008; Ader et al. 2009a, b; Schneider et al. 2008).
Remaining intensities at 10.92 kHz in the spectral regime [40, 25 ppm] can be well explained by transfer across short, mostly intra-residue distances. The identified aromatic-aliphatic side-chain correlations are compatible with the detection of sequential side-chain contacts between L86 and W87 (green box) and long range contacts between W68 (Cζ2, Cζ3, Cη2) and A47Cβ. Furthermore, we could identify resolved intermonomer contacts between the aromatic-aliphatic side-chains (highlighted in red) as well as cross peaks between specific amino acid pairs (F–A Cβ, F–T/V Cγ1/2) that solely encode intermolecular contacts (red boxes).
Overall, our results on a fractionally deuterated ion channel embedded in lipid bilayers suggest a reduction in molecular protonation by approximately 50% in line with earlier solution-state NMR work on globular proteins (Rosen et al. 1996; Shekhtman et al. 2002; Otten et al. 2010). Reminiscent of effects seen using specifically-labeled glycerol precursors during bacterial growth (LeMaster and Kushlan 1996; Hong 1999; Castellani et al. 2002), the exact deuteration level can vary in protein side-chains and methyl positions are expected to contain several isotopomers. In 1H correlation experiments, we observed an improved spectral dispersion and a reduction in line width compared to the protonated case. In standard PDSD experiments on [1H/2H,13C,15N] KcsA-Kv1.3, one-bond (13C,13C) correlations appeared after 20 ms mixing similar to fully protonated KcsA-Kv1.3 suggesting that polarization transfer dynamics are not strongly affected by the reduction in proton density. However, additional studies will be needed to dissect in detail the polarization transfer rates as a function of MAS rate, B0 field and local proton density. In general, our experiments were conducted at modest MAS rates (10-14 kHz), it seems likely that further improvements in 1H line width and spectral resolution may be possible using a combination of dedicated pulse schemes (Sakellariou et al. 2000), ultra-fast MAS (Wickramasinghe et al. 2009; Bertini et al. 2010b) and ultra-high field NMR systems.
The reduced protonation level also simplifies the spectral analysis of 13C/15N correlation experiments. Because residual protons are preferably found at the amino-acid side-chains (Fig. 7 insert, Supporting Information table 1), polarization transfer among and spectroscopic assignments of protein side-chain positions is facilitated. For long mixing times and longitudinal mixing schemes, proton-mediated transfer becomes band-selective around the rotational resonance conditions among aliphatic, aromatic and carboxyl carbons. Experimental results shown here suggest that these conditions can aid the detection of medium to long-range correlations occurring in a particular spectral window. Notably, such measurements also revealed intermolecular contacts in our tetrameric [1H/2H,13C,15N] ion channel for which the combined application of dedicated ssNMR schemes and mixed labelling approaches that previously allowed detecting such constraints (see, e.g., Etzkorn et al. 2004; Wasmer et al. 2008; Etzkorn et al. 2010) is precluded. It seems likely that fractional deuteration will also facilitate the determination of longer internuclear distances using rotational-resonance recoupling (Spencer et al. 1991; Costa et al. 1997) or rotating-frame (Nomura et al. 1999; Sonnenberg et al. 2004) and MAS-modulated variants (Verel et al. 1997; Ramachandran et al. 2003) thereof. In addition, coherent transfer schemes that mediate (13C,15N) transfer via proton spins such as CHC (Seidel et al. 2005), PAR (Paepe et al. 2008) or PAIN-CP (De Paepe et al. 2011) experiments may be readily combined with fractional deuteration to suppress chemical-shift offset affects or to enhance transfer efficiencies.
Compared to schemes involving (partially) deuterated precursors, fractional deuteration reduces the influence of isotope effects on ssNMR chemical shifts (Hansen 1988) and offers a cost efficient way to sizably reduce protonation levels in complex biomolecules. These considerations and our results suggest that fractional deuteration can provide a powerful means to aid structural studies of complex biomolecules by high-resolution ssNMR.
The research leading to these results has received funding from the European Community’s Seventh Framework Programme (FP7/2007-2013) under grant agreement number 261863. Financial support from NWO is gratefully acknowledged (grants 700.26.121 and 700.58.102). We thank Karin Giller for technical support.
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