Conformational features and ionization states of Lys side chains in a protein studied using the stereo-array isotope labeling (SAIL) method
Although both the hydrophobic aliphatic chain and hydrophilic ζ-amino group of the Lys side chain presumably contribute to the structures and functions of proteins, the dual nature of the Lys residue has not been fully investigated using NMR spectroscopy, due to the lack of appropriate methods to acquire comprehensive information on its long consecutive methylene chain. We describe herein a robust strategy to address the current situation, using various isotope-aided NMR technologies. The feasibility of our approach is demonstrated for the Δ+PHS/V66K variant of staphylococcal nuclease (SNase), which contains 21 Lys residues, including the engineered Lys-66 with an unusually low pKa of ∼ 5.6. All of the NMR signals for the 21 Lys residues were sequentially and stereospecifically assigned using the stereo-array isotope-labeled Lys (SAIL-Lys), [U-13C,15N; β2,γ2,δ2,ε3-D4]-Lys. The complete set of assigned 1H, 13C, and 15N NMR signals for the Lys side-chain moieties affords useful structural information. For example, the set includes the characteristic chemical shifts for the 13Cδ, 13Cε, and 15Nζ signals for Lys-66, which has the deprotonated ζ-amino group, and the large upfield shifts for the 1H and 13C signals for the Lys-9, Lys-28, Lys-84, Lys-110, and Lys-133 side chains, which are indicative of nearby aromatic rings. The 13Cε and 15Nζ chemical shifts of the SNase variant selectively labeled with either [ε-13C;ε,ε-D2]-Lys or SAIL-Lys, dissolved in H2O and D2O, showed that the deuterium-induced shifts for Lys-66 were substantially different from those of the other 20 Lys residues. Namely, the deuterium-induced shifts of the 13Cε and 15Nζ signals depend on the ionization states of the ζ-amino group, i.e., −0.32 ppm for Δδ13Cε [NζD-NζH] vs. −0.21 ppm for Δδ13Cε [NζD2-NζH2] and −1.1 ppm for Δδ15Nζ[NζD-NζH] vs. −1.8 ppm for Δδ15Nζ[NζD2-NζH2]. Since the 1D 13C NMR spectrum of a protein selectively labeled with [ε-13C;ε,ε-D2]-Lys shows narrow (> 2 Hz) and well-dispersed 13C signals, the deuterium-induced shift difference of 0.11 ppm for the protonated and deprotonated ζ-amino groups, which corresponds to 16.5 Hz at a field strength of 14 T (150 MHz for 13C), could be accurately measured. Although the isotope shift difference itself may not be absolutely decisive to distinguish the ionization state of the ζ-amino group, the 13Cδ, 13Cε, and 15Nζ signals for a Lys residue with a deprotonated ζ-amino group are likely to exhibit distinctive chemical shifts as compared to the normal residues with protonated ζ-amino groups. Therefore, the isotope shifts would provide a useful auxiliary index for identifying Lys residues with deprotonated ζ-amino groups at physiological pH levels.
Detailed studies on the structures and dynamics of the Lys residues in a protein have been severely hampered by the difficulty in gathering comprehensive NMR information on their side-chain moieties. It is especially challenging to establish unambiguous stereospecific assignments for the prochiral protons in the four consecutive methylene chain, which is the longest aliphatic chain among the 20 common amino acids. Given the lack of generally applicable strategies to overcome this obstacle, only a few NMR studies have probed the structural aspects of stereospecifically assigned Lys residues. The ionization states of the Lys ζ-amino groups also provide important information, as they are often involved in specific intra- and/or intermolecular molecular recognition processes and thus play vital roles in protein functions. Therefore, the side-chain moieties of Lys residues contribute to maintaining the structure and biological functions of a protein by two elements: the hydrophobic methylene chain and the hydrophilic ζ-amino group. To investigate this dual nature of the Lys side chain, we have applied various isotope-aided NMR technologies, including the stereo-array isotope labeling (SAIL) method (Kainosho et al., 2006).
The Lys ζ-amino groups, which usually have pKa values around 10.5, are protonated (NH) at around neutral pH. However, certain proteins have Lys residues with deprotonated ζ-amino groups, even at neutral or acidic pH (Harris and Turner, 2002). In such cases, the pKa values of the Lys ζ-amino group are substantially lowered owing to its particular local environment. Since the Lys ζ-NH2 groups are endowed with significantly different physical chemical properties, as compared to the ζ-NH, they can perform specific functions, such as Schiff base formation through nucleophilic attacks on various substrates (Highbarger et al., 1996; Barbas et al., 1997). Although the ionization states of Lys ζ-amino groups in a protein have been inferred from X-ray crystallographic maps, they are subject to misinterpretation and may not always be identical to those in solution. NMR spectroscopy provides methods for determining the charge state of Lys side chains. For example, the NH and NH2 states of Lys residues in solution can be identified from cross-peak patterns in the 1H–15N correlation NMR spectra, if the hydrogen exchange rates are sufficiently slow, or from the values of 15Nζ and/or 1Hζ chemical shifts (Poon et al., 2006; Iwahara et al., 2007; Takayama et al., 2008). Under physiological conditions, however, the observations of 1H–15N cross peaks are often hampered due to the rapid hydrogen exchange rates of the Lys ζ-amino groups (Liepinsh et al., 1992; Liepinsh and Otting, 1996; Otting and Wüthrich, 1989; Otting et al., 1991; Segawa et al., 2008). The ionization states can also be identified by the pH titration profiles for the 13Cε and 15Nζ signals of individual Lys residues (Kesvatera et al., 1996; Damblon et al., 1996; Farmer and Venters, 1996; Poon et al., 2006; Gao et al., 2006; André et al., 2007). Unfortunately, long-term experiments such as pH titrations are hampered by the stability and solubility issues of a protein over the required pH range. Therefore, straightforward and robust alternative methods to identify Lys residues with distinct ionization states for the ζ-amino groups are highly desired.
We used a variant of staphylococcal nuclease, Δ+PHS/V66K SNase (denoted as the SNase variant hereafter), as the model protein (Stites et al., 1991). This variant was engineered to add the following three features to the wild-type SNase: (i) introduction of three stabilizing mutations, P117G, H124L, and S128A (PHS); (ii) deletion of amino acids 44–49 and introduction of two mutations, G50F and V51N (Δ); and (iii) substitution of Val66 with Lys (V66K). With these three modifications, the Δ+PHS/V66K SNase variant becomes thermally stable, even with the ζ-amino group of Lys-66 entrapped within the hydrophobic cavity originally occupied by the Val-66 side chain in the wild-type SNase. As a result, the ζ-amino group of Lys-66 in the SNase variant exhibits an unusually low pKa value of 5.7 (García-Moreno et al., 1997; Fitch et al., 2002).
Although the SNase variant contains 21 Lys residues (Fig. A1), including the engineered Lys-66, the 13C, 1H, and 15N NMR signals for the Lys side chains were unambiguously observed and assigned using the SNase variant selectively labeled with SAIL-Lys, i.e., L-[U-13C,15N; β2,γ2,δ2,ε3-D4]-Lys (Kainosho et al., 2006; Terauchi et al., 2011). In this article, we examine some of the structural features inferred from the comprehensive chemical shift data and the deuterium-induced isotope shifts on the 13Cε and 15Nζ of the Lys residues in the SNase variant and show that the side-chain NMR signals can serve as powerful probes to investigate the dual nature of a Lys side chain in a protein.
2.1 Sample preparation
The Δ+PHS/V66K SNase variants selectively labeled with either L-[U-13C,15N]-Lys, L-[U-13C,15N; β2,γ2,δ2,ε3-D4]-Lys (SAIL-Lys), or L-[ε-13C;ε,ε-D2]-Lys, which were synthesized in-house, were prepared using the E. coli BL21 (DE3) strain transformed with a pET3 vector (Novagen), encoding the Δ+PHS/V66K SNase gene fused with an N-terminal His-tag. The transformed E. coli cells were cultured at 37 ∘C in 500 mL of M9 medium, containing anhydrous Na2HPO4 (3.4 g L−1), anhydrous KH2PO4 (0.5 g L−1), NaCl (0.25 g L−1), D-glucose (5 g L−1), NH4Cl (0.5 g L−1), thiamine (0.5 mg L−1), FeCl3 (0.03 mM), MnCl2 (0.05 mM), CaCl2 (0.1 mM), and MgSO4 (1 mM), with 10 mg L−1 of the monohydrochloride salts of either [U-13C,15N]-Lys, SAIL-Lys, or [ε-13C;ε,ε-D2]-Lys. Each culture was maintained at 37 ∘C. An additional 20 mg L−1 of each isotope-labeled Lys was supplemented when the OD600 reached 0.5, and then protein expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM. At 4–5 h after the induction, the cells were collected by centrifugation, and the SNase variant proteins were purified on a Ni-NTA column (Isom et al., 2008). The enrichment levels for Lys were ∼ 70 %, as measured using mass spectrometry. The purified proteins were dissolved in 20 mM sodium phosphate buffers containing 100 mM KCl (pH 8.0), together with a small amount of DSS as the internal chemical shift reference, prepared with either H2O, D2O, or H2O : D2O (1 : 1). The chemical shifts for 1H, 13C, and 15N were primarily referenced to the methyl proton signal of the internal DSS according to the IUPAC recommendation (Markley et al., 1998). However, we usually convert the δDSS (1H 13C) to δTSP (1H 13C) simply by adding 0.15 ppm, i.e., δDSS–δTSP = 0.15 ppm, for adjustment to the previous δTSP–(1H) chemical shifts reported for wt (wild-type) SNase (Torchia et al., 1989). On the other hand, all of the 15N chemical shifts are referenced to DSS, to facilitate the chemical shift comparison with the recent 15N data (Takayama et al., 2008). The chemical shift references are mentioned in the footnotes of the figures and tables.
2.2 NMR spectroscopy
The 600 MHz 2D 1H–13C constant-time heteronuclear single quantum coherence correlation (HSQC) spectra of the SNase variant, selectively labeled with either [U-13C,15N]-Lys or SAIL-Lys, were measured in D2O at 30 ∘C on a Bruker Avance spectrometer equipped with a TXI cryogenic probe. For the latter sample, additional deuterium decoupling was applied during the t1 period. The data sizes and spectral widths were 1024 (t1) × 2048 (t2) points and 12 000 Hz (ω1, 13C) × 8700 Hz (ω2, 1H), respectively. Each set of 32 scans per free induction decay was collected with a 1.5 s repetition time, using the 13C carrier frequency at 38 ppm. The 600 MHz 3D HCCH total correlation spectroscopy (TOCSY) spectrum was measured in D2O at 30 ∘C for the SNase variant labeled with SAIL-Lys (Clore et al., 1990; Cavanagh et al., 2007). The data size and spectral width were 1024 (t1) × 32 (t2) × 2048 (t3) points and 6000 Hz (ω1, 1H) Hz × 9100 Hz (ω2, 13C) × 9000 Hz (ω3, 1H), respectively. Each set of 16 scans/FID with a 1.5 s repetition time was collected, using the 13C carrier frequency at 40 ppm.
The Lys ζ-15N signals of the SAIL-Lys-labeled SNase variant dissolved in D2O at 30 ∘C were assigned using the HECENZ pulse sequence, utilizing the out-and-back magnetization transfer from 1Hε2 to 15Nζ via 13Cε. The correlations between the 1Hε2 and 15Nζ signals for most of the 21 Lys residues were firmly established by the pulse sequence, which was basically the same as the H2CN pulse sequence developed by André et al. (2007). The data size and the spectral width were 512 (t1) × 1024 (t2) points and 1200 Hz (ω1, 15N) Hz × 9600 Hz (ω2, 1H), respectively, and deuterium decoupling was applied during the t1 period. The carrier frequencies were 38 and 28 ppm for 13C and 15N, respectively, and 128 scans/FID with a 2 s repetition time were accumulated.
The 125.7 MHz 1D 13C NMR spectra of the SNase variant proteins selectively labeled with either [U-13C,15N]-Lys or [ε-13C;ε,ε-D2]-Lys were measured in D2O, H2O, and H2O : D2O (1 : 1), at 25 ∘C on a Bruker Avance 500 spectrometer equipped with a DCH cryogenic probe; simultaneous deuterium decoupling was achieved using the WALTZ16 scheme. The spectral width and repetition time were 6300 Hz and 5 s, respectively. In the experiment in H2O solution, a 4.1 mm o.d. Shigemi tube containing the protein solution was inserted into a 5 mm o.d. outer tube containing pure D2O for the internal lock signal. By taking advantage of the selective deuteration on the ε-13C in [ε-13C;ε,ε-D2]-Lys (∼ 98 at. %), the background 13C signals due to the naturally abundant, and therefore protonated, 13C nuclei were readily filtered out using the pulse scheme shown in Fig. A2.
3.1 Complete assignment of the Lys side-chain NMR signals in the SNase variant selectively labeled with SAIL-Lys
Although the chemical shifts with sequential assignments for the backbone 1H, 13C, and 15N signals of SNase are available in the BMRB (entry #16123; Chimenti et al., 2011), we reconfirmed them through the HNCA experiment for the [U-13C, 15N]-SNase variant, since the solution conditions were slightly different. The complete side-chain assignment for all 21 Lys residues was not trivial, even for the SNase variant residue selectively labeled with [U-13C, 15N]-Lys, due to the extensive signal overlap as illustrated in the F1–F3 projection of the 3D HCCH TOCSY spectrum (Fig. 1a). On the other hand, a markedly improved 3D HCCH TOCSY spectrum was obtained, under the simultaneous deuterium decoupling, for the SNase variant residue selectively labeled with SAIL-Lys (Fig. 1b), enabling us to firmly establish the full connectivity for the side-chain 1H, 13C, and15N NMR signals of the 21 Lys residues. To illustrate the improved spectral quality obtained with the SAIL-Lys in lieu of [U-13C,15N]-Lys, a panel obtained for the F1–F3 projection, along the 13C axis (F2) restricted for the chemical shift range of 40.1–45.5 ppm for the 13Cε signals, is shown for the 1Hα–1Hε2 correlation signals (Fig. 1c). By taking advantage of the well-dispersed 1Hα–1Hε2 signals, the backbone 1Hα–13Cα signals (Fig. 1e) were readily correlated to the 1Hε2–13Cε HSQC signals (Fig. 1d). Actually, all of the SAIL-Lys side-chain 13C signals were facilely and unambiguously assigned through the 3D HCCH TOCSY spectrum, yielding a complete set of the Lys side-chain NMR chemical shifts, as summarized in Table 1. It should be noted that since each one of the SAIL-Lys side-chain methylene groups (–CHD–) was stereospecifically deuterated, i.e., [U-13C,15N; β2,γ2,δ2,ε3-D4]-Lys (Fig. 1f), the Lys β3, γ3, δ3, and ε2-1H signals of the side chains of the 21 Lys residues were stereospecifically assigned. Thus, these assigned signals have the potential of providing a wealth of information on the local conformations of the Lys side chains in solution.
3.2 Structural information inferable from the Lys side-chain chemical shifts
Note that the chemical shifts in Table 1 for the 21 Lys residues in the SAIL-Lys-labeled SNase variant are not corrected for the various isotope-induced shifts caused by the complicated isotope-labeling pattern of the SAIL-Lys structure (see Fig. 1f). Based on comprehensive NMR data, we should be able to elucidate the dual role of the Lys side chains in terms of the conformational dynamics and functional properties of a protein in further detail, using various solution NMR methods. In this section, we briefly interpret the chemical shift data to characterize the local conformational features by the 1H, 13C, and 15N signals compiled in Table 1, which should be followed by more extensive studies in the future. Although we have not yet attempted to collect the comprehensive nuclear Overhauser effects (NOEs), such as using a fully SAIL-labeled SNase variant (Kainosho et al., 2006), it was obvious that the chemical shift data with exclusive and unambiguous assignments for the Lys residues contain an abundance of information on the side-chain conformations and ionization states of the ζ-amino groups. As described above, the unusual chemical shifts of the Lys-66 side chain confirmed the deprotonated state of its ζ-amino group. We also obtained some interesting structural information for the other Lys residues with protonated ζ-amino groups. For example, the Lys-9 side chain exists in two conformational states in the crystalline state (PDB entry #3HZX), which only differ in the χ4 angle, i.e., Form A (trans, ∼ −175∘) and Form B (gauche+, ∼ +44∘), as shown in Fig. 2a and b, respectively (see also Table A1). The significantly upfield-shifted signals observed for Lys-9 relative to the averaged chemical shifts (Δδ, ppm) are obviously due to the aromatic ring current of Tyr-93, i.e., 15Nζ (30.8 ppm, ppm), 13CHε2 ( ppm, ppm), and 1Hδ3 (1.04 ppm, ppm). These chemical shifts suggest the ζ–NH3+–π interaction, as shown by the dashed red line (Fig. 2a). Therefore, the chemical shifts for Lys-9 strongly imply that the van der Waals interactions between the aliphatic side chain, as well as the electrostatic interaction between the positively charged ζ-HN3+ and the nearby aromatic ring of Tyr-93, simultaneously contribute to preferentially stabilize the Form A conformation in solution (Fig. 2a).
The upfield shifts of the side-chain methylenes, induced by the neighboring aromatic rings, were also detected for Lys-28, Lys-84, Lys-110, and Lys-133. Considering the local structures of Lys-28 and Lys-84 in the crystal (Fig. 2c, d), the relative orientations between Lys-28 and Tyr-27 and between Lys-84 and Tyr-85 seem to be similar to those in the crystal and are responsible for the large upfield shifts for only their 1Hγ3 signals, i.e., Lys-28: 0.61 ppm, ppm; Lys-84: 0.64 ppm, ppm, while the other 13C and 1H shifts remain within the average ranges (Table 1). The small but obvious low-field shifts for the 15Nζ (Lys-28: 31.9 ppm; Lys-84: 32.0 ppm; Δδ: +0.3 and 0.4 ppm, respectively) might be caused by the electrostatic interactions between the Oη of Tyr-27 Tyr-85 and the Nζ of Lys-28 Lys-84, respectively, as shown by the dashed red lines (Fig. 2c, d). The bulky indole ring of Trp-140 seems to simultaneously stabilize the aliphatic chains of both Lys-133 and Lys-110, inducing the upfield shifts for some of the side-chain signals, i.e., Lys-133 13CHε2 (40.9 2.10 ppm, ppm), 1Hδ3 (1.15 ppm, ppm), 1Hγ3 (0.59 ppm, ppm) and 1Hβ3 (1.42 ppm, ppm); Lys-110 1Hε2 (2.68 ppm, ppm). These upfield shifted signals indicate that the van der Waals interactions between the methylene moieties of Lys-133 and Lys-110, with the hydrophobic indole ring of Trp-140 sandwiched in the middle, are also preserved in solution (Fig. 2e). Interestingly, the chemical shift differences between the two prochiral protons attached to the ε-carbons, observed for the SNase variant residue selectively labeled with [U-13C,15N]-Lys, of Lys residues 110 and 133 are considerably larger than those of the other 19 Lys residues, which are much smaller than ∼ 0.05 ppm (Fig. A3). Since the 1Hε2 chemical shifts were observed at a 0.15 and 0.17 ppm higher field than the 1Hε3 chemical shifts for Lys-110 and -133, respectively, the conformations of these two Lys residues are likely to be similar to those in the crystal (Fig. 2e).
On the other hand, the unusual chemical shifts observed for the Lys-66 residue, which is trapped within the hydrophobic environment engineered in the engineered SNase variant (Fig. 2f), clearly reveal the strong influence of the ionization state of the ζ-amino group on the Lys side chain. As shown in Table 1, the 15Nζ chemical shift of the ζ-ND2 of Lys-66 in the SNase variant appears at an unusually upfield position, as compared to the averaged chemical shift range for the ζ-ND3+ in the other Lys residues, i.e., 15Nζ (Lys-66: 20.9 ppm, ppm), which is close to the value of the ζ-NH2 chemical shift, 23.3 ppm, previously reported for Lys-66 in the [U-13C,15N]-SNase variant (André et al., 2007; Takayama et al., 2008). Evidently, the 15Nζ chemical shifts provide an unambiguous clue to distinguish between the deprotonated and protonated ζ-amino groups of Lys residues. However, the complete side-chain assignment including the terminal ζ-15N signals through conventional methods using a [U-13C,15N] protein is usually laborious and occasionally not practical.
In comparison with charged Lys side chains, deprotonation of the ζ-amino group of Lys-66 is characterized by sizable 1H and 13C chemical shift differences, i.e., 13CHε2 ( ppm, ppm), 13CHδ3 ( ppm, ppm), and 13CHγ3 ( ppm, ppm). These deprotonation shifts, in particular, those of 13Cε and/or 13Cδ, could therefore be used as unambiguous indices to characterize the ionization states of the ζ-amino groups of Lys residues in a protein, since they can be accurately and readily observed and assigned using a protein selectively labeled with SAIL-Lys. It should be noted, however, that the side-chain chemical shifts in general might significantly vary according to the local environments, such as the relative position to aromatic rings, and thus the results obtained exclusively from the side-chain chemical shifts might not be absolutely reliable. To avoid any possible uncertainties in characterizing the ionization states of ζ-amino groups, an alternative approach using the deuterium-induced isotope shifts of the SAIL-Lys side-chain 13C signals may be considered.
3.3 Characterization of the ionization state of the ζ-amino group of Lys residues using the effects of deuterium-induced isotope shifts on the side-chain 13C and 15N signals
In our previous studies investigating the effects of the deuterium-induced isotope shifts on the 13C signals adjacent to polar functional groups with an exchangeable hydrogen, such as hydroxyl (OH) or sulfhydryl (SH) groups, we demonstrated that those isotope shifts are versatile indices for identifying residues, such as Tyr, Thr, Ser, or Cys, with exceptionally slow hydrogen exchange rates (Takeda et al., 2014). For example, in a protein selectively labeled with [ζ-13C]-Tyr, the Tyr residues have much slower hydrogen exchange rates for the η-hydroxyl groups than the isotope shift differences in the 13Cζ signals and exhibit well-resolved pairwise signals with nearly equal intensities in the 1D 13C NMR spectrum in H2O : D2O (1 : 1) (Takeda et al., 2009). The up- and low-field counterparts of the pairwise 13Cζ signals correspond to those in D2O and H2O, respectively, and their relative intensities reflect the fractionation factors, i.e., [OD] [OH]. Similar approaches have been developed for Ser, Thr, and Cys residues, using the 13Cβ signals observed for proteins selectively labeled with [β-13C; β, β-D2]-Ser, [β-13C; β-D]-Thr, and [β-13C; β, β-D2]-Cys, respectively (Takeda et al., 2010, 2011). Since the isolated 13Cβ(D2) or 13Cβ(D) moieties in the labeled amino acids give extremely narrow signals under the deuterium decoupling, the 13C NMR signals can be obtained with remarkably high sensitivities, especially with a 13C direct observing cryogenic probe. Interestingly, while the fractionation factors for the Ser and Thr hydroxyl groups, i.e., [OD] [OH], are usually close to unity, as also for the Tyr residues, those for the Cys sulfhydryl groups, i.e., [SD] [SH], are around 0.4–0.5 (Takeda et al., 2010, 2011). The methods are especially important, since the functional groups of the residues readily identified as having exceptionally slow hydrogen exchange rates are most likely to be involved in hydrogen bonding networks and/or located in distinctive local environments.
Although the idea of estimating the hydrogen exchange rates by the deuterium-induced isotope shifts on the 13C nuclei adjacent to functional groups with exchangeable hydrogens was originally exploited years ago, for the backbone amide groups in the selectively labeled proteins with [C'-13C]-amino acid(s) (Kainosho and Tsuji, 1982; Markley and Kainosho, 1993), it has not yet been applied for the Lys ζ-amino groups. Having established the complete assignment for the 21 Lys residues in the SNase variant selectively labeled with SAIL-Lys (Table 1), we next examined the deuterium-induced chemical shifts in detail for the Lys side-chain signals. In the case of Lys residues, the NMR signals of the ζ-amino 15N and ε- or δ-carbon 13C signals would be plausible candidates for probing the deuterium substitution effects. There have several reports on the isotope shifts of the δ- and ε-13C for the Lys residues induced by the deuteration of ζ-amino groups (Ladner et al., 1975; Led and Petersen, 1979; Hansen, 1983; Dziembowska et al., 2004; Tomlinson et al., 2009; Platzer et al., 2014). However, it seems no comprehensive studies have applied the deuterium-induced isotope shifts on 13Cε signals to characterize the ionization states of Lys residues.
We first examined the 1D 13C and 15N NMR spectra of [15N2]-Lys in D2O and H2O, at pH 8 and 30 ∘C, to choose the suitable NMR probes to distinguish between the deprotonated and protonated ζ-amino groups (Fig. 3). The ζ-15N signal appears at ∼ 1 ppm upfield in D2O relative to that in H2O (Fig. 3a), and the aliphatic 13C signals of [15N2]-Lys at the natural abundance also showed isotope shifts, Δδ[13Ci (in D2O)-δ13Ci (in H2O)], i.e., 13Cα, −0.25 ppm; 13Cβ, −0.20 ppm; 13Cγ, −0.03 ppm; 13Cδ, −0.17 ppm; and 13Cε, −0.31 ppm (Fig. 3b). Although the isotope shifts for 13Cα and 13Cβ are due to the deuteration of the α-amino group, those for 13Cδ and 13Cε are obviously due to the deuteration of the ζ-amino group. Considering the finding that the 13Cε of Lys gives an isolated signal far from the others and exhibits a ∼ 1.8-fold larger isotope shift as compared to 13Cδ, the 13Cε and 15Nζ signals seem to be good candidates for probing the ionization states of Lys residues in the SNase variant.
Although the 15Nζ and 13Cε chemical shifts for the Lys residues can be measured by the HECENZ and 1H–13C ct (constant time) HSQC experiments, respectively, using the SNase variant selectively labeled with [U-13C,15N]-Lys or SAIL-Lys, it was rather difficult to determine the accurate isotope shifts of the 15Nζ and 13Cε signals for all 21 Lys residues using these methods. In particular, the accurate chemical shift measurement for an individual 13Cε signal was hampered by the poor quality of the ct HSQC spectrum, even for the protein labeled with SAIL-Lys (Fig. A3). Therefore, we used [ε-13C; ε,ε-D2]-Lys to reduce the line widths of the 13Cε signals for the Lys residues in the SNase variant. As expected, the 1D 13C NMR spectra of the SNase variant selectively labeled with [ε-13C;ε,ε-D2]-Lys showed remarkably well-resolved signals, with line widths less than 2 Hz, under the 1H 2D double decoupling conditions (Fig. 4). Note that the weak background signals due to the naturally abundant 13C nuclei were filtered out in this spectrum (Fig. A2). By referring to the chemical shifts in Table 1, which were determined using the 3D HCCH TOCSY experiment for the SNase labeled with SAIL-Lys, all of the 1-D 13Cε signals were unambiguously assigned (Fig. 4a, b). The chemical shifts of 13Cε are slightly different among the data sets, because the isotope shifts induced by the nearby isotopes on the 13Cε signals are different for SAIL-Lys and [ε-13C;ε,ε-D2]-Lys (Tables 1, 2). The 13Cε chemical shifts in H2O and D2O, which were accurately determined by the 1D 13C NMR spectra, are presented in Fig. 5. At a glance, the 13Cε spectra in Fig. 5a and c look almost the same, since the signals moved upfield with a constant increment of ppm, except for the 13Cε signal of Lys-66 (Table 2). Since the δ13Cε values in H2O and D2O are very close to those for the free [15N2]-Lys (Fig. 3b), the ζ-amino groups are protonated in H2O and deuterated in D2O, and thus the averaged deuterium-induced isotope shift was designated as Δδ13Cε [NζD-NζH]. Similarly, the averaged Δδ15Nζ [NζD-NζH], excluding the value for Lys-66, was determined to be ppm (Williamson et al., 2013), which was also close to the free [15N2]-Lys (Fig. 3a). The Δδ13Cε and Δδ15Nζ for Lys-66, which are −0.21 and −1.8 ppm (Table 2), respectively, confirmed that the ζ-amino group of this residue is deprotonated at pH 8 and should be designated as Δδ13Cε [NζD2-NζH2] and Δδ15Nζ [NζD2-NζH2]. Interestingly, the fact that the averaged Δδ13Cε [NζD-NζH], −0.32 ppm, was ∼ 1.5 times larger than the Δδ13Cε [NζD2-NζH2] for Lys-66, −0.21 ppm, might suggest that the deuterium-induced isotope shift on 13Cε is proportional to the number of hydrogen atoms on the ζ-amino groups. In contrast, the averaged Δδ15Nζ [NζD-NζH], −1.1 ppm, was much smaller than that of the Δδ15Nζ [NζD2-NζH2] for Lys-66, −1.8 ppm.
We also measured the 1D 13C NMR spectrum of the SNase variant selectively labeled with [ε-13C;ε,ε-D2]-Lys in H2O : D2O (1 : 1), to search for the Lys residues with slowly exchanging ζ-amino groups. Clearly, there are no such residues in the SNase variant at pH 8 and 30 ∘C, as shown in Fig. 5b. Due to the rapid hydrogen exchange rates for all 21 Lys residues in this protein, the observed isotope shifts on 13Cε were exactly half of the Δδ13Cε [NζD2-NζH2] for Lys-66 or Δδ13Cε [NζD-NζH] for the rest of the Lys residues. The hydrogen exchange rate constant (kex) for the ζ-amino group of Lys-66 in the SNase variant, which is deeply embedded in the hydrophobic cavity originally occupied by Val-66 in the wild-type SNase, was 93±5 s−1 at pH 8 and −1 ∘C (Takayama et al., 2008). Therefore, the hydrogens on the ζ-amino groups in all 21 Lys residues in the SNase variant are rapidly exchanging, and thus the observed chemical shifts for the 13Cε of Lys-66 and the rest of the Lys residues in H2O : D2O (1 : 1) are the time averages for three isotopomers, NH2, NHD, and ND2, with nearly a 1 : 2 : 1 ratio for Lys-66, and for four isotopomers, NH, NH2D+, NHD, and ND, with a ratio of 1 : 3 : 3 : 1. Since the time-averaged signals for Lys-66 and other Lys residues in H2O : D2O (1 : 1) appeared exactly in the middle of the spectra observed in H2O and D2O (Fig. 5a, c), the fractional factors for the isotopomers are nearly identical, as statistically random distributions.
In this article, we have shown that comprehensive NMR information can be obtained using cutting-edge isotope-aided NMR technologies for the Lys side-chain moieties, comprising a long hydrophobic methylene chain and a hydrophilic ζ-amino group, to facilitate hitherto unexplored investigations toward elucidating the dual nature of the Lys residues in a protein. The unambiguously assigned 13C signals, together with the stereospecifically assigned prochiral protons for each of the long consecutive methylene chains, which first became available using the stereo-array isotope labeling (SAIL) method, provide unprecedented opportunities to examine the conformational features around the Lys residues in detail. The ionization states of the ζ-amino groups of Lys residues, which play crucial roles in the biological functions of proteins, could be readily characterized by the deuterium-induced isotope shifts on the ε-13C signals observed by the 1D 13C NMR spectroscopy of a protein selectively labeled with [ε-13C;ε,ε-D2]-Lys. Both methods should work equally well for larger proteins, for which previous NMR approaches were rarely applicable. Therefore, these methods will contribute toward clarifying the structural and functional roles of the Lys residues in biologically important proteins.
All of the NMR data supporting this work are shown in the paper. Isotopically labeled lysines are available on request from Taiyo Nippon Sanso Co. at https://stableisotope.tn-sanso.co.jp (last access: 23 April 2021).
MT prepared the isotopically labeled protein samples, collected and analyzed NMR data, and wrote the paper. YM measured and analyzed high-field NMR spectra. TT prepared isotopically labeled lysines. MK supervised the project and edited the paper.
The authors declare that they have no conflict of interest.
This article is part of the special issue “Robert Kaptein Festschrift”. It is not associated with a conference.
This work was performed, in part, using the NMR spectrometers with the ultra-high magnetic fields under the Collaborative Research program of the Institute for Protein Research, Osaka University (grant nos. NMRCR-19-05 and NMRCR-20-05).
This research has been supported by Grants-in-Aid in Innovative Areas (grant nos. 21121002 and 26119005) and also in part by the Kurata Memorial Hitachi Science and Technology Foundation and by Grants-in-Aid for Scientific Research (grant no. 25440018).
This paper was edited by Rolf Boelens and reviewed by three anonymous referees.
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