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. Author manuscript; available in PMC: 2015 Aug 1.
Published in final edited form as: J Magn Reson. 2014 Jun 26;245:105–109. doi: 10.1016/j.jmr.2014.06.005

Differentiation of Histidine Tautomeric States using 15N Selectively Filtered 13C Solid-State NMR Spectroscopy

Yimin Miao 1, Timothy A Cross 1,2, Riqiang Fu 2,*
PMCID: PMC4136442  NIHMSID: NIHMS613744  PMID: 25026459

Abstract

The histidine imidazole ring in proteins usually contains a mixture of three possible tautomeric states (two neutral - τ and π states and a charged state) at physiological pHs. Differentiating the tautomeric states is critical for understanding how the histidine residue participates in many structurally and functionally important proteins. In this work, one dimensional 15N selectively filtered 13C solid-state NMR spectroscopy is proposed to differentiate histidine tautomeric states and to identify all 13C resonances of the individual imidazole rings in a mixture of tautomeric states. When 15N selective 180° pulses are applied to the protonated or non-protonated nitrogen region, the 13C sites that are bonded to the non-protonated or protonated nitrogen sites can be identified, respectively. A sample of 13C,15N labeled histidine powder lyophilized from a solution at pH 6.3 has been used to illustrate the usefulness of this scheme by uniquely assigning resonances of the neutral τ and charged states from the mixture.

Keywords: Histidine tautomeric states, solid-state MAS NMR, REDOR/TEDOR, selective polarization

1. Introduction

Histidine is an essential residue in many proteins [1, 2]. With its imidazole ring serving as a general base when it is neutral and serving as a general acid when in its charged form, it participates in enzyme catalyzed reactions, assists in stabilizing protein structures, mediates proton transfer and so on. The imidazole side chain has a pKa of approximately 6, which means it plays active roles at physiologically relevant pHs, where a small shift in pH will affect protonation of the imidazole ring. When it acts as a proton shuttle, the basic nitrogen on its imidazole ring can take up a proton to form a charged intermediate, then give up a proton to a solvent molecule before returning to the uncharged state, potentially shuttling a proton from one side of a membrane to the other. For instance, the set of four histidine side chains in the pore of the tetrameric M2 protein from Influenza A virus is the heart of H+ conductance responsible for H+ selectivity, pH activation and interactions with the W41 gate through the protonation and depotonation of the H37 imidazole rings [3, 4]. Therefore, differentiating the tautomeric states is critical for understanding how the histidine residue plays its structurally and functionally important roles in proteins.

There are three possible tautomeric states for histidine (Scheme 1). At higher pH values, two neutral tautomers can exist: the τ tautomer with non-protonated Nδ1 and protonated Nε2, and the π tautomer with protonated Nδ1 and non-protonated Nε2. While at lower pH values, the imidazole ring becomes charged with both Nδ1 and Nε2 protonated. At physiological pH values different tautomeric states often coexist providing an important indication of the biological processes that are taking place in a system. NMR spectroscopy is an effective approach for detecting different tautomeric states of histidine. However, in biological systems, the situation is complex -both 13C and 15N chemical shifts of the histidine residue are very sensitive to its tautomeric states, hydrogen bond lengths, the chemical environment of the imidazole rings and the backbone structure of the protein. Indeed, the 13C chemical shift range tabulated in the BioMagRes Databank has a range of 17 ppm for the Cγ site, 48 ppm for the Cδ2 site and 40 PCM for the Cε1 site. Moreover, the resonance linewidths are highly dependent on the dynamic and proton exchange properties of the imidazole rings and consequently signals from different tautomers can be severely overlapped making it almost impossible to identify different tautomeric states based on the known benchmarks from the histidine amino acid [5-7]. Here, we propose 15N selectively filtered 13C NMR spectroscopy to uniquely differentiate histidine tautomeric states in a uniformly 13C and 15N labeled histidine sample at pH 6.3.

Scheme 1.

Scheme 1

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Since the neutral states have their unique non-protonated nitrogen sites (Nδ1 for τ tautomer and Nε2 for π tautomer), whose chemical shifts are different from that of the protonated nitrogen, 15N NMR is a common approach for calculating the relative amount of neutral and charged histidines. However, there are two intrinsic shortcomings associated with the 15N NMR analyses. First, cross polarization (CP) efficiency. It is known that CP [8, 9] is an essential NMR technique in solid state NMR used to enhance the sensitivity of dilute spins like 15N. However, the dipolar coupling between the protonated nitrogen and its bonded proton is much stronger than that between the non-protonated nitrogen and its closest proton, so that the CP dynamics for the protonated and non-protonated nitrogen are rather different. As a result, the magnetization for the protonated nitrogen can be built up very quickly, while the signals for the non-protonated nitrogen require a much longer CP contact time to buildup. In light of short proton spin relaxation time in the rotating frame (i.e. T1ρ), which is commonly observed in many proteins, it is rather inefficient to polarize the non-protonated nitrogen signals. Although spin-locking the proton magnetization along the magic angle can lengthen the T1ρ value by a factor of two or three to improve the CP efficiency [10], it is necessary to perform CP variable contact time experiments to analyze the CP dynamics in order to accurately quantitate the protonated and non-protonated nitrogen signals [10]. However, when the population of the neutral tautomeric states is low, it is difficult to observe the non-protonated nitrogen signals [3] even if the CP contact time is long. Secondly, the resonances may overlap. For the neutral tautomeric states, the protonated nitrogen also exist (Nε2 for τ tautomer and Nδ1 for π tautomer). These protonated nitrogen signals may overlap with the nitrogen signals from the charged tautomer. Therefore, it is necessary to subtract the protonated nitrogen signals contributed from neutral tautomers in order to accurately calculate the total signals from the charged tautomer, which is an impossible task with current one dimensional (1D) spectra. Two-dimensional (2D) heteronuclear 15N-13C correlation spectra are required, often combined with other correlation spectra such as 13C-13C correlations, to make resonance assignments. Such experiments are time-consuming. Site specific labeled histidine has to be used to differentiate signals from Nε2 and Nδ1, which is costly.

In this paper, we propose nitrogen selectively filtered 13C NMR spectroscopy to differentiate tautomeric states of histidine. In NMR, 13C is known to be more sensitive than 15N. Therefore, observing 13C instead of 15N provides much needed sensitivity for detecting different tautomers, while protonated or non-protonated nitrogens are used to select different 13C signals. More importantly, the CP dynamics for each carbon site in the histidine tautomers is similar, so that there is no concern about CP efficiency for seeing different tautomeric states. The advantages of this method will be demonstrated by using a histidine powder sample lyophilized from a solution at pH 6.3.

2. Materials and methods

2.1. Preparation of histidine powder sample

Uniformly 13C, 15N-labeled L-Histidine•HCl•H2O, pH 3.3 was purchased from Cambridge Isotope Laboratories, Inc. The crystalline histidine was dissolved in deionized water and titrated with 0.2 M NaOH to reach pH 6.3, then lyophilized in the vacuum pump. The lyophilized histidine powder was packed into a 3.2mm MAS rotor.

2.2. NMR spectroscopy

All MAS NMR spectra were acquired on a Bruker Avance 600.1 MHz NMR spectrometer using a NHMFL 3.2 mm Low-E triple-resonance biosolids MAS probe [11, 12]. The sample spinning rate was controlled within 13.5kHz±3Hz by a Bruker pneumatic MAS unit. The 13C magnetization was enhanced through CP from protons with a contact time of 1 ms, during which a 1H spin-lock field of 50 kHz was used and the 13C B1 field was ramped from 38 to 56 kHz [13]. The 13C and 15N 180° pulse lengths were experimentally calibrated to be 6.6 and 7.9 μs, respectively. The 15N selective 180° pulse was achieved using an 1-side lobe Sinc shape with a pulse-length of 280 μs. A SPINAL64 decoupling sequence [14] with a 1H B1 field of 78 kHz was used for proton decoupling. The 13C chemical shifts were referenced to the carbonyl carbon resonance of glycine at 178.4 ppm relative to 4,4-dimethyl-4-silapentanesulfonate sodium (DSS), and the 15N chemical shifts reference was calculated by the known relative frequency ratios between DSS (13C) and liquid ammonia (15N) [15, 16].

2.3. Experimental strategy

Table 1 lists the observable 13C resonances of different histidine tautomeric states that are expected under various 1D experiments to be performed. We will explain in detail in the next section how the 13C resonances from each tautomer can be differentiated based on these 1D experiments.

Table 1. 13C Peaks for Histidine Tautomers Observed under Various Experiments.

Experiment being performed graphic file with name nihms613744t1.jpg graphic file with name nihms613744t2.jpg graphic file with name nihms613744t3.jpg
13C-1H dipolar dephasing Cγ Cγ Cγ
15NALL-filtering Cγ, Cδ2, Cε1 Cγ, Cδ2, Cεl Cγ, Cδ2, Cεl
15NH-filtering Cδ2, Cε1 Cγ, Cεl Cγ, Cδ2, Cε1
15NnonH-filtering Cγ, Cεl Cδ2, Cε1 N/A
Double Quantum Filtering Cγ, Cδ2 Cγ, Cδ2 Cγ, Cδ2
15NnonH-filtering 13C-13C Cγ, Cεl, {Cδ2} Cδ2, Cεl, {Cγ } N/A
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As shown in Scheme 1, each tautomer exhibits three carbons (Cγ, Cδ2, and Cε1) and two nitrogens (Nδ1 and Nε2). Since Cγ is the quaternary carbon that does not have a directly bonded proton, its dipolar interaction with its closest proton is considerably weaker compared to the tertiary carbons (Cδ2, and Cε1) that have directly bonded protons. Thus a simple 13C-1H dipolar dephasing experiment [17] can be performed to differentiate Cγ from Cδ2 and Cε1. Since the traditional dipolar dephasing scheme becomes less efficient in terms of suppressing the protonated carbon signals under fast spinning rate, an efficient rotational-echo double resonance (REDOR) [18] based dipolar dephasing scheme is shown in Fig. 1a, while the double-quantum filtering (DQF) [19, 20] experiment can be used to select for directly bonded 13C-13C pairs identified using a short filtering time. Consequently, Cγ and Cδ2 resonances for all tautomers are identified.

Fig. 1.

Fig. 1

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Pulse sequences used in our experiments. (a) REDOR based dipolar dephasing sequence; (b) REDOR based nitrogen selective dephasing sequence; (c) 15N selective filtering 13C spectroscopy. Δ and Δ′ are set to one rotor period during which the 1H rf amplitude of ωr is applied for effective z-filtering. When Δ′ is long, the 13C-13C transfers are facilitated via spin diffusion under the dipolar assisted rotational resonance (DARR) condition. In all experiments, Tr represents the spinning period, while τmix is the total mixing time for TEDOR. The phase cycles were as follows: (a) ϕ1 = 02, ϕ2 = 0022 1133, ϕ3 = 0, ϕrec = 0220 3113; (b) ϕ1 = 02, ϕ2 = 0022 1133, ϕ3 = 8×(0) 8×(1) 8×(2) 8×(3), ϕ4 = 02, ϕrec = 0220 3113 2002 1331; (c) ϕ1 = 16×(0) 16×(2), ϕ2 = 0, ϕ3 = 0, ϕ4 = 0213 2031, ϕ5 = 2, ϕ6 = 0, ϕ7 = 02, ϕ8 = 1133, ϕ9 = 4×(0) 4×(1) 4×(2) 4×(3), ϕrec = 3113 0220 1331 2002 1331 2002 3113 0220, where 0 = x, 1 = y, 2 = -x, and 3 = -y. All 180° pulses during REDOR/TEDOR were phase cycled using xy-4.

Fig. 1b shows the pulse sequence for 15N dephased 13C NMR experiments. Without the 15N selective 180° pulse, the 13C-15N dipolar couplings averaged by MAS will be reintroduced by a train of 15N 180° hard pulses synchronized with MAS, resulting in dephasing of 13C signals, as explained in REDOR [18]. However, when the 15N selective pulse is applied to the nonprotonated 15N (or 15NnonH), the dipolar couplings between 13C and 15NnonH will not be reintroduced because of the additional 180° flip of the 15N magnetization, so that the 13C signals will only be dephased by the protonated 15N (or 15NH), not by 15NnonH. Similarly, when the 15N selective pulse is applied to 15NH, the 13C signals will be dephased by 15NnonH, not by 15NH.

The pulse sequence used for 15N selective filtering in 13C MAS NMR experiments is shown in Fig. 1c. Without the 15N selective 180° pulses, this sequence (Fig. 1c) is the 1D version of the z-filtered transferred-echo double resonance (TEDOR) sequence for 15NALL-filtered 13C spectra [21]. In the histidine imidazole ring, the bond length between the bonded C-N pairs is about 1.4 Å, while the next nearest C-N distance (i.e. Nδ1-Cδ2 and Nε2-Cγ) is about 2.2 Å. As indicated in the z-filtered TEDOR spectra [21], at ∼1 ms TEDOR mixing time the covalently bonded NC cross peaks have maximum signals while the Nδ1-Cδ2 and Nε2-Cγ cross peaks generate weak signals because of their longer distances. Therefore, by using ∼1ms mixing time, we can effectively select for the directly 15N bonded 13C resonances without polarizing the carbons opposite to the nitrogen sites in the imidazole ring.

When the selective 180° 15N pulses are applied to the non-protonated nitrogen region (∼250 ppm), the magnetization for the non-protonated nitrogen will be additionally inverted so that any dipolar interactions between the non-protonated nitrogen and carbons will be refocused, leading to diminished signals for these carbons. The protonated nitrogens (in the region of ∼180 ppm) are not affected by these selective pulses and hence the signals for the carbons directly bonded to the protonated nitrogens remain. Similarly, the selective 180° 15N pulses applied to the protonated nitrogen region will suppress the protonated nitrogen bonded carbons while allowing the non-protonated bonded carbon resonances to be observed.

3. Results and discussion

Fig. 2 shows the aromatic regions of 13C MAS NMR spectra of a histidine powder sample lyophilized from a pH 6.3 solution. For each histidine tautomeric state, three 13C resonances are expected. Six 13C peaks (139.5, 138.2, 137.3, 130.2, 121.2, and 115.4 ppm) observed in the traditional CPMAS spectrum (c.f. Fig. 2a) suggest that there exist in this preparation two different histidine tautomers at pH 6.3. The spectrum with a 1 ms mixing time shows relatively uniform spectral intensity for the protonated and non-protonated 13C sites. While in the 15N solid-state CPMAS NMR spectrum shown in Fig. 3a, the non-protonated 15N peak at 248.7 ppm is much smaller than the other protonated resonances between 160 and 190 ppm at this given CP contact time (3 ms) owing to different CP efficiencies. Due to its high gyromagnetic ratio, 13C has a much better CP transfer from protons, as compared with 15N, even for carbon sites that are not directly proton-bonded. Fig. 2b shows the REDOR based dipolar dephasing 13C spectrum using the pulse sequence in Fig. 1a. Two peaks at 139.5 and 130.2 ppm remain while the other peaks are completely dephased. Thus the two peaks at 139.5 and 130.2 ppm are the quaternary Cγcarbons. The DQF spectrum in Fig. 2c shows an additional two resonances at 121.2 and 115.4 ppm besides the Cγ resonances. These two new peaks can be assigned to the Cδ2 carbons which are bonded to Cγ, while Cε1 is bonded to Nδ1 and Nε2. However, none of the carbon resonances have yet to be assigned to specific histidine tautomeric states.

Fig. 2.

Fig. 2

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13C NMR spectra in the aromatic region of the histidine powder lyophilized from a pH 6.3 solution. (a) CPMAS with 8 scans; (b) REDOR based dipolar dephasing spectrum with 8 scans and m=2; (c) DQF spectrum with 32 scans.

Fig. 3.

Fig. 3

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(a) 15N CPMAS NMR spectrum of the histidine powder lyophilized from a pH 6.3 solution. 3 ms CP contact time was used, during which the 1H B1 field of 50 kHz was used and the 15N B1 field was ramped from 44 to 63 kHz. 16 scans were used to accumulate the signals. REDOR based 15N dipolar dephasing 13C spectrum (b) without and (c) with a 15N selective 180° pulse applied to the protonated 15N region at 180 ppm with m=1 and τmix = 2.08 ms. 8 scans were used to accumulate the signals.

Fig. 3b shows the REDOR based 15N-13C dipolar dephasing 13C spectra using the pulse sequence in Fig. 1b without the 15N selective 180° pulse. Due to the dipolar recoupling, all the 13C resonances are effectively dephased by near-by 15N. However, when the 15N selective 180° pulse is applied to the protonated region at 180 ppm, the protonated 15N magnetization experiences an additional 180° flip in the z-axis so that the dipolar coupling generated by the REDOR sequence is refocused. As a result, there is no dephasing for those carbons that are bonded to the protonated 15N. Since the 15N selective 180° pulse does not affect the non-protonated 15N sites, the recoupled dipolar interaction between the carbons and non-protonated nitrogens by REDOR remains so that these 13C (-NnonH) sites continue to be effectively dephased. Thus by varying the power level of the shaped pulse, we can maximize the signals from the carbons that are bonded with the protonated nitrogen so as to precisely determine the 15N selective 180° pulse. As shown in Fig. 3c, the resonances at 139.5 and 137.3 ppm are greatly dephased, while the resonances at 138.2, 130.2, 121.2, and 115.4 ppm remain almost intact. Therefore, by applying the 15N selective 180° pulse, we should be able to differentiate the 13C (-NnonH) from the 13C (-NH) sites.

The key to the success for this type of selective filtering experiments is 15N selectivity. Fig. 4 shows an array of 13C spectra in the region between 142 and 135 ppm as a function of offset for the 15N selective 180° pulse. The resonance at 138.2 ppm belongs to a carbon bonded with a protonated 15N (Cε1-NH) while the peaks at 139.5 (Cγ-Nδ1nonH) correspond to carbons bonded with a non-protonated 15N. The offset at 0 Hz was centered on the protonated 15N region (180 ppm), while the negative offsets are shifted towards the non-protonated nitrogen frequencies. With the 15N selective 180° pulse width of 280 μs, the signal intensity at 138.2 ppm is about 90% and 60% when the 15N offset is ±1000 and ±2000 Hz, respectively, as compared to that at the offset center. On the other hand, the 15N dipolar dephased 13C peak at 139.5 ppm decreases in intensity as the 15N offset is shifted further away from the non-protonated 15N region. Therefore, the bandwidth for the 15N selective 180° pulse is sufficient to cover the protonated chemical shift range, which is about 40 ppm, corresponding to 2400 Hz on the 600 MHz NMR spectrometer, while it has little effect on the non-protonated 15N, which is more than 60 ppm (i.e. > 3600 Hz on the 600 MHz) away from the protonated 15N region.

Fig. 4.

Fig. 4

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Experimental 15N selective dipolar dephasing 13C spectra (showing only the region between 142 and 135 ppm) as a function of 15N offset. In this plot, the 15N offset at 0 Hz corresponds to the middle of the protonated 15N region (180 ppm), while the negative offset is shifted towards the non-protonated region (250 ppm). The 13C resonances at 138.2 ppm (∼ at the center of each spectrum) and at 139.5 ppm, which have a small and large dephasing effect, respectively, by the 15N selective 180° pulse, are used to demonstrate the selectivity of the given selective 180° pulse.

Fig. 5a shows the 15NALL-filtered 13C spectrum for the aromatic region of the histidine powder sample using the pulse sequence diagramed in Fig. 1c without the 15N selective 180° pulses. Since all of the covalently linked 15N-13C pairs are recoupled, the 15nall filtered 13C spectrum has a similar resonance pattern to that of the CPMAS spectrum in Fig. 2a, with all six 13C resonances observed from the two different histidine tautomers present in this sample. When the 15N selective 180° pulses are applied to the non-protonated region at 250 ppm, the sequence generates the 15NH-filtered 13C spectrum as shown in Fig. 5b. According to Table 1, for the 15NH filtered 13C spectrum, it is expected to have two resonances from each of the neutral states and three resonances from the charged state. The fact that there are five 13C resonances in Fig. 5b suggests that there is a charged tautomer in this histidine sample.

Fig. 5.

Fig. 5

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15N filtered 13C spectra of the imidazole rings for the histidine powder at pH 6.3 using the pulse sequence in Fig. 1c. (a) 15NALL-filtered 13C spectrum with no 15N selective 180° pulses applied. (b) 15NH-filtered 13C spectrum with the 15N selective 180° pulses applied to the non-protonated 15N region at 250 ppm. (c) 15NnonH-filtered 13C spectrum with the 15N selective 180° pulses applied to the protonated 15N region at 180 ppm. (d) The same as in (c) but having a long Δ′ (=4 ms) to permit 13C-13C transfer. In all experiments, τmix=1.185 ms, m=1, Δ=74.07 μs, and 32 scans were used to accumulate the signals.

When the selective 180° pulses are applied to the protonated 15N region at 180 ppm, the sequence produces the 15NnonH-filtered 13C spectrum as shown in Fig. 5c. In this spectrum, no resonances from a charged state should appear. Fig. 5c shows two resonances, one of which represents Cγ at 139.5 ppm, as indicated in the dipolar dephased 13C spectrum in Fig. 2b. Therefore, this signal at 139.5 ppm can be unambiguously assigned to Cγ of the τ tautomer, while the other resonance at 137.3 ppm must be Cε1 of the τ tautomer. By comparing Fig. 2b and Fig. 5b, the other Cγ peak at 130.2 is assigned to the charged tautomer. Since the two peaks at 121.2 and 115.4 ppm are assigned to Cδ2 (c.f. Fig. 2), the signal at 138.2 ppm in Fig. 5b can be assigned to Cε1 of the charged tautomer.

So far we have assigned Cγ and Cε1 to their respective τ and charged tautomers. However, we have not yet assigned the two Cδ2 resonances to their specific tautomers. In the Fig. 1c pulse sequence, there are two z-filters. When the second z-filter is set to a longer time such as Δ′ to 4 ms, an extra transfer between 13C's will take place either through 13C-13C spin diffusion under the dipolar assisted rotational resonance (DARR) condition [22, 23] or 13C-13C chemical exchange. Fig. 5d shows the 15NnonH-filtered 13C spectrum using Δ′ = 4 ms. Clearly, the signal at 115.4 ppm starts to appear, indicating that this resonance must be Cδ2 of the τ tautomer, since no carbon signal from the charged histidine is expected to go through the 15NnonH-filter.

At high pH, the two neutral τ and π states may coexist. Differentiating their resonances is straightforward and unambiguous by a simple subtraction of the 15NH- from 15NnonH-filtered 13C spectra, provided that their respective resonances from the neutral τ and π states do not overlap. As indicated in Table 1, Cδ2 and Cε1 of the τ tautomer and Cγ and Cε1 of the π tautomer are expected to appear in the 15NH-filtered 13C spectrum, while Cγ and Cε1 of the τ tautomer and Cδ2 and Cε1 of the π tautomer are observable in the 15NnonH-filtered 13C spectrum. As the Cε1 signals derive from one bond Cε1-Nδ1 or Cε1-Nε2 in both the 15NH- and 15NnonH-filtering experiments, the difference between the 15NH- and 15NnonH-filtered 13C spectra will completely suppress the Cε1 resonances from the τ and π states. This gives rise to a positive Cδ2 resonance and a negative Cγ peak for the τ tautomer, and results in a negative Cδ2 resonance and a positive Cγ peak for the π tautomer.

4. Conclusion

It has been demonstrated experimentally that one-dimensional 15N selective filtered 13C NMR spectroscopy provides an efficient way to differentiate histidine neutral and charged states by identifying the 13C sites bonding with the protonated or non-protonated nitrogen in the imidazole ring of histidine. Unlike the spectral editing methods [24] that were designed to assign the 13C and 15N resonances of histidine imidazole ring in a single tautomeric state, the new 15N selective filtering techniques proposed here, along with dipolar dephasing and DQF experiments, are able to identify all 13C resonances of individual histidine imidazole ring from mixed tautomeric states. Therefore, this proposed methodology opens up a new way of investigating histidine pH titration processes in functionally important biological systems.

Highlights.

  • 1D 15N filtered 13C ssNMR spectroscopy is proposed to differentiate histidine tautomers

  • 15N selective pulses are used to choose non-protonated and protonated bonded 13C sites

  • All 13C resonances of individual imidazole rings from mixed tautomers can be identified

  • This may open up a new way of investigating histidine pH titration processes in proteins

Acknowledgments

This work was supported by NIH Grant AI23007. All NMR experiments were carried out at the National High Magnetic Field Lab (NHMFL) supported by the NSF Cooperative agreement N. DMR-1157490 and the State of Florida.

Footnotes

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