Determination of Histidine Protonation States in Proteins by Fast Magic Angle Spinning NMR

Histidine residues play important structural and functional roles in proteins, such as serving as metal-binding ligands, mediating enzyme catalysis, and modulating proton channel activity. Many of these activities are modulated by the ionization state of the imidazole ring. Here we present a fast MAS NMR approach for the determination of protonation and tautomeric states of His at frequencies of 40–62 kHz. The experiments combine 1H detection with selective magnetization inversion techniques and transferred echo double resonance (TEDOR)–based filters, in 2D heteronuclear correlation experiments. We illustrate this approach using microcrystalline assemblies of HIV-1 CACTD-SP1 protein.


INTRODUCTION
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).
Herein, we present an alternative MAS experiment that uses 1 H detected transferred-echo double resonance (TEDOR)based 15 N selectively filtered 2D correlations at fast MAS frequencies of 40-60 kHz. The advantages of the 1 H-detected fast-MAS experiments presented here are: i) improved sensitivity due to 1 H detection, and ii) improved resolution via the second dimension and selective recoupling of aromatic resonances directly attached to 15 N atoms. Microcrystalline assemblies of U-13 C, 15 N-and fractionally deuterated (FD) (Mance et al., 2015) 13 C, 15 N-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.

Sample Preparation
U-13 C, 15 N-L-histidine was purchased from Cambridge Isotope Laboratories, recrystallized from an aqueous solution at pH 6.0, adjusted by mixing HCl and NaOH. The sample was packed into a 1.3 mm MAS rotor. Microcrystalline assemblies of U-13 C, 15 N-and FD-13 C, 15 N-HIV-1 CA CTD -SP1 were prepared in the presence of the assembly cofactor inositol hexakisphosphate (IP6) as described previously (Wagner et al., 2016) except for growing Escherichia coli in M9 medium containing 13 C glucose, 15 N NH 4 Cl, isotopically labeled precursors, and (for the deuterated sample) D 2 O. Proteins were assembled with 1.6 mM IP6 (Sigma-Aldrich), for a final reaction volume of 1 ml at pH 8.0. Assemblies were incubated overnight at 20°C and packed into 3.2 mm (U-13 C, 15 N), 1.9 mm (FD-13 C, 15 N), or 1.3 mm MAS rotors (U-13 C, 15 N).

MAS NMR Spectroscopy
MAS NMR experiments on U-13 C, 15 N-CA CTD -SP1 and FD-13 C, 15 N-CA CTD -SP1 microcrystalline assemblies were performed on a 20.0 T Bruker AVIII spectrometer outfitted with 3.2 mm E-Free HCN and 1.9 HCN probes, respectively. The MAS frequency was 14 and 40 kHz, respectively, controlled to within ± 10 Hz by a Bruker MAS controller. The actual sample temperature was maintained at 4 ± 1°C throughout the experiments using the Bruker temperature controller.
The Larmor frequencies were 850.4 MHz ( 1 H), 213.9 MHz ( 13 C) and 86.2 MHz ( 15 N). The typical 90°pulse lengths were 2.6-3.0 μs for 1 H, 4.3-4.5 μs for 13 C, and 4.2-4.7 μs for 15 N. The 1 H-13 C and 1 H-15 N cross-polarization employed a linear amplitude ramp of 90-110% on 1 H, and the center of the ramp was matched to a Hartmann-Hahn condition at the first spinning sideband; contact times of 0.7-1.5 ms and 1.0-1.7 ms were used, respectively. 50 ms CORD (Hou et al., 2013) mixing time was applied to facilitate 13 C-13 C mixing.
MAS NMR experiments on U-13 C, 15 N-L-histidine and FD-13 C, 15 N-CA CTD -SP1 microcrystalline assemblies were performed on a 14.1 T Bruker AVIII spectrometer outfitted with 1.3 mm HCN probe. Larmor frequencies were 599.8 MHz ( 1 H), 150.8 MHz ( 13 C), and 60.7 MHz ( 15 N). The MAS frequency was 60 kHz, controlled to within ± 10 Hz by a Bruker MAS controller. The actual sample temperature was maintained at 40 ± 1°C throughout the experiments using the Bruker temperature controller. The typical 90°pulse lengths were 1.4-1.6 μs for 1 H, 2.7-3.0 μs for 13 C, and 3.3-3.6 μs for 15 N. The 1 H-13 C and 1 H-15 N cross-polarization employed a linear amplitude ramp of 90-110% on 1 H, center of the ramp was matched to a Hartmann-Hahn condition at the first spinning sideband, with contact times of 1.0-5.0 ms and 1.3-5.0 ms, respectively. Band-selective 15 N-13 C SPECIFIC-CP contact time was 5.0-6.0 ms. SWFTPPM (Vinod Chandran et al., 2008) decoupling (15 kHz) was used during the TEDOR block and acquisition periods. The selective 15 N 180°r-SNOB (Kupce et al., 1995) pulse length in the Z-filtered TEDOR experiments was 500 µs and the bandwidth -2 kHz; the rf power was 4 kHz. During the Z-filter time period Δ, 60 kHz CW decoupling was applied for τ r on 1 H channel, while during the time period Δ', MISSISSIPPI (Zhou and Rienstra, 2008) water suppression was applied. The TEDOR block duration was 1-3 ms.

Data Processing
All MAS NMR data were processed using NMRPipe (Delaglio et al., 1995). The 13 C and 15 N chemical shifts were referenced with respect to the external standards adamantane (Morcombe and Zilm, 2003) and ammonium chloride (Bertani et al., 2014), respectively. The 2D and 3D data sets were processed by applying 30, 45, 60, and 90°shifted sine bell apodization followed by a Lorentzian-to-Gaussian transformation in both dimensions. Forward linear prediction to twice the number of the original data points was used in the indirect dimension followed by zero filling. The processed spectra were analyzed in NMRFAM-Sparky (Goddard and Kneller, 2004;Lee et al., 2015) and CCPN (Stevens et al., 2011).

RESULTS
Here, we report on a 2D 1 H-detected TEDOR-based Z-filtered experiment, which incorporates 15 N 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) 15 N selective TEDOR filter, containing 13 C resonances of all protonation and tautomeric states present; ii) 15 N 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) 15 N 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 15 N 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 13 C and 15 N 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 13 C-detected TEDOR-based experiments are well suited for the determination of protonation states in this sample. To test the 1 H-detected sequences proposed herein, three complementary experiments were performed. As shown in Figure 1G, 15 N 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 15 N 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-13 C, 15 N-CA CTD -SP1 sample allows for the determination of histidine protonation and tautomeric states even in the 13 C-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 15 N selective TEDOR-filtered 13 C 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 13 C-13 C 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 1 H-detected TEDORbased 15 N selective CH HETCOR spectra ( Figure 2D) also indicate the presence of a single species, which is unambiguously assigned as τ tautomer. These 1 H-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-13 C, 15 N-CA CTD -SP1, the His-226 protonation state in U-13 C, 15 N-CA CTD -SP1 assemblies cannot be easily determined using the 1D 13 C-detected version of TEDOR-based 15 N selective filtered experiments due to low resolution and spectral overlap ( Figure 3A). In contrast, the 2D 1 H-detected TEDOR-based 15 N 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 1 H-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-13 C, 15 N -CA CTD -SP1 and can be distinguished from those corresponding to the histidine based on chemical shift.

CONCLUSION
We demonstrated that 1 H-detected 2D Z-filtered TEDOR experiments incorporating 15 N 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.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

AUTHOR CONTRIBUTIONS
TP and AMG conceived the project and guided the work. RZ performed NMR experiments and analyzed the experimental data. CMQ assisted with the NMR experiments and data analysis. SS assigned the CA CTD -SP1 chemical shifts. KKZ, BKG-P, and OP prepared samples of microcrystalline CA CTD -SP1 assemblies. RZ and TP took the lead in writing the manuscript. All authors discussed the results and contributed to the manuscript preparation.

FUNDING
This work was supported by the National Institutes of Health (NIH Grants P50AI1504817 and R01AI129678). We acknowledge the support of the National Science Foundation (NSF Grant CHE0959496) for the acquisition of the 850 MHz NMR spectrometer and of the National Institutes of Health (NIH Grant P30GM110758) for the support of core instrumentation infrastructure at the University of Delaware; and of the National Institutes of Health (NIH Grant S10OD012213) for the acquisition of the 750 MHz NMR spectrometer at the University of Pittsburgh.