Histidines (His) play important structural and functional roles in proteins such as metal binding (Stryer et al., 1964; Perutz and Mathews, 1966; Adams et al., 1969; Liljas et al., 1972), proton transfer (Hoffee et al., 1967; Blow et al., 1969; Campbell et al., 1974), and stability (Perutz et al., 1969; Lewis et al., 1976; Loewenthal et al., 1992). These functions are often correlated with the ionization state of the histidine sidechain (Figure 1A) (Bachovchin and Roberts, 1978; Kossiakoff and Spencer, 1981; Lewis et al., 1981). While the pK_a of the imidazole ring for free histidine is 6.5 (Blomberg et al., 1977), in proteins the pK_a values vary widely, from 3 to 9, depending on the interactions with neighboring residues and degree of burial (Zhou et al., 1993; Plesniak et al., 1996). At pH values above the pK_a, anionic τ and π tautomers with hydrogens at either N^ε2 or N^δ1 are present, while below the pK_a the protonated imidazole ring possesses hydrogens at both N^ε2 and N^δ1. For a protein at intermediate pH values, it is possible that a fraction of His residues is protonated and the remaining fraction unprotonated (French and Hammes, 1965; Edwards and Sykes, 1980; Hass et al., 2008).

Methods to determine His ionization states in proteins are solution NMR (Kilmartin et al., 1973; Markley, 1975; Bachovchin and Roberts, 1978; Perutz et al., 1985; Pelton et al., 1993; Shimba et al., 1998; Hass et al., 2008; Hansen and Kay, 2014) or neutron diffraction (Kossiakoff and Spencer, 1980; Maeda et al., 2004; Kovalevsky et al., 2010), with the latter limited to very large single crystals and requiring a neutron source, both difficult conditions to meet routinely. Therefore, solid-state magic angle spinning (MAS) NMR constitutes a viable alternative (Wei et al., 1999). Similar to solution NMR, the tautomeric state of histidines can be unambiguously determined from a unique combination of ¹⁵N sidechain chemical shifts (Munowitz et al., 1982; Wei et al., 1999; Miao et al., 2014) and the corresponding N-H distances can be estimated, allowing for hydrogen bonding studies (Shenderovich et al., 2015). Protonation states for the crystalline histidine amino acid have been determined by MAS NMR for different pH values (Li and Hong, 2011) and crystalline short peptides (Platzer et al., 2014). Using ¹⁵N selective filtered, ¹³C-detected experiments with the inversion pulses at frequencies of the different tautomers (Miao et al., 2014) permits their identification. For proteins containing several histidine residues, the above experiments are challenging due to low sensitivity and spectral overlap. Therefore, only a handful of such studies have been reported to date (Hu et al., 2006; Hu et al., 2010; Miao et al., 2015; Kwon et al., 2019; Maciejko et al., 2019; Vasa et al., 2019; Movellan et al., 2020). In order to increase resolution, the original pulse sequence can be reconfigured as a 2D experiment by introducing a ¹³C-¹³C mixing period based on proton-driven spin diffusion (PDSD) (Bloembergen, 1949) and extending the second Z-filter (Miao et al., 2014). 2D and 3D proton-based experiments were also introduced with ¹H chemical shifts either recorded in the indirect dimension (Miao et al., 2015) or detected directly (Shenderovich et al., 2015; Vasa et al., 2019; Movellan et al., 2020).

Herein, we present an alternative MAS experiment that uses ¹H detected transferred-echo double resonance (TEDOR)-based ¹⁵N selectively filtered 2D correlations at fast MAS frequencies of 40–60 kHz. The advantages of the ¹H-detected fast-MAS experiments presented here are: i) improved sensitivity due to ¹H detection, and ii) improved resolution via the second dimension and selective recoupling of aromatic resonances directly attached to ¹⁵N atoms. Microcrystalline assemblies of U-¹³C,¹⁵N- and fractionally deuterated (FD) (Mance et al., 2015) ¹³C,¹⁵N-HIV-1 CA_CTD-SP1 protein samples, possessing solely a single His residue, His-226, are ideally suited for pulse sequence optimization and therefore were selected for illustrating our current approach. Extension to ultrafast MAS frequencies (up to 110 kHz), should yield even higher sensitivity and resolution for proteins with multiple histidines.

Here, we report on a 2D ¹H-detected TEDOR-based Z-filtered experiment, which incorporates ¹⁵N selective filters for the determination of histidine tautomeric states. The pulse sequence is shown in Figure 1B. The experiment is well suited for fast MAS frequencies of 40 kHz and above. The tautomeric states of His residues are unambiguously determined using a combination of three CH HETCOR experiments comprising: i) ¹⁵N selective TEDOR filter, containing ¹³C resonances of all protonation and tautomeric states present; ii) ¹⁵N selective TEDOR filter with a soft pulse at 170 ppm, removing resonances of the protonated state while C^ε1 and C^δ2 atoms of π tautomer and C^ε1 and C^γ atoms of τ tautomer remain; and iii) ¹⁵N selective TEDOR filter with a soft pulse at 250 ppm, retaining all signals of the charged state, C^ε1 and C^γ of the π tautomer as well as C^ε1 and C^δ2 atoms of the τ tautomer. C^ε1 of anionic tautomers is always present in TEDOR filtered spectra, but has reduced peak intensity when ¹⁵N selective pulse is applied as C-N dipolar interaction with the non-selectively irradiated nitrogen atom is recoupled. The sequence was first tested on a crystalline L-histidine sample prepared at pH 6.0. The ¹³C and ¹⁵N 1D CPMAS and 2D NCA spectra are shown in Figures 1D, E, respectively. The spectra clearly indicate the presence of two forms of L-histidine, the charged monohydrate and the τ tautomer, in approximately 2:1 ratio. As shown in Figure 1F, conventional ¹³C-detected TEDOR-based experiments are well suited for the determination of protonation states in this sample. To test the ¹H-detected sequences proposed herein, three complementary experiments were performed. As shown in Figure 1G, ¹⁵N selective TEDOR-filtered CH HETCOR without or with a soft pulse at 250 ppm (left and right panels, respectively) yield the sidechain signals of both protonation states, while ¹⁵N selective TEDOR-filtered CH HETCOR with soft pulse at 170 ppm retains only C^ε1 resonance of the τ tautomer (chemical shifts provided in Supplementary Table S1). Water suppression was incorporated into the second Z-filter, allowing to record spectra on hydrated samples.

HIV-1 CA_CTD-SP1 (Figure 2A) contains a single His residue, His-226. The outstanding high spectral resolution in the microcrystalline FD-¹³C,¹⁵N-CA_CTD-SP1 sample allows for the determination of histidine protonation and tautomeric states even in the ¹³C-detected mode (Figure 2B). The C^ε1 and C^γ resonances are present in 1D experiments, while the C^δ2 resonance is absent in the ¹⁵N selective TEDOR-filtered ¹³C CPMAS experiment with the soft pulse at 170 ppm since its magnetization does not build up during the TEDOR block due to the very weak dipolar coupling to N^δ1 (chemical shifts provided in Supplementary Table S2). The 2D ¹³C-¹³C CORD spectrum clearly shows a single set of resonances, indicating the presence of only one histidine species (Figure 2C), although the protonation and tautomeric state cannot be determined without additional experiments. The three complementary ¹H-detected TEDOR-based ¹⁵N selective CH HETCOR spectra (Figure 2D) also indicate the presence of a single species, which is unambiguously assigned as τ tautomer. These ¹H-detected 2D spectra contain no resonances of aromatic residues other than His (shown in black in the CH HETCOR spectrum) and Trp (these are weak or absent in the spectra of the deuterated sample), as only carbons attached to nitrogens are selected, making assignment of histidine resonances straightforward.

In contrast to the FD-¹³C,¹⁵N-CA_CTD-SP1, the His-226 protonation state in U-¹³C,¹⁵N-CA_CTD-SP1 assemblies cannot be easily determined using the 1D ¹³C-detected version of TEDOR-based ¹⁵N selective filtered experiments due to low resolution and spectral overlap (Figure 3A). In contrast, the 2D ¹H-detected TEDOR-based ¹⁵N selective filtered spectra (Figure 3B) suggest the presence of a small fraction of π tautomer along with the predominant τ tautomer in this sample (chemical shifts provided in Supplementary Table S2).

In addition to the His signals, the indole ring signals of the Trp184 residue are also present in the ¹H-detected TEDOR-based experiments when the soft pulse is either turned off or centered at 250 ppm. This is expected due to the nitrogen atom N^ε1 in the indole ring, which allows for magnetization build up on adjacent carbon atoms (C^δ1 and C^ε2) during TEDOR transfer. Tryptophan sidechain resonances appear much stronger in non-deuterated protein assemblies compared to the FD-¹³C,¹⁵N -CA_CTD-SP1 and can be distinguished from those corresponding to the histidine based on chemical shift.

We demonstrated that ¹H-detected 2D Z-filtered TEDOR experiments incorporating ¹⁵N selective filters permit unambiguous assignment of histidine protonation and tautomeric states in microcrystalline proteins and protein assemblies. This approach combines all the advantages of fast MAS and proton detection. Extending the experiments to MAS frequencies of 110 kHz and above can further improve the quality of data sets and allow unambiguous assignment of His protonation and tautomeric states in larger proteins and protein assemblies.