Nuclear magnetic resonance spectroscopy

In 1945, first radio-frequency signals of the nuclei of atoms could be measured and observed (Purcell et al., 1946; Bloch et al., 1946). This event denotes the birth of nuclear magnetic resonance spectroscopy. NMR is based on the principle of the alignment of atomic nuclei with a magnetic moment in a strong, homogenous magnetic field. Directed radio-frequency pulses allow these nuclear spins to be tilted out of their equilibrium position. During the return to the equilibrium state, referred as relaxation, the nuclei emit signals with their own resonance frequencies. NMR enables their detection and interpretation. Resonance frequencies of NMR-active nuclei are strongly dependent on the chemical surrounding. The environment like solvent molecules, amino acid side chains, bound inhibitors and many more affect the actual magnetic field to which each individual nucleus is exposed. Hence, the

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resonance frequency of these nuclei shifts. The value of this chemical shift in ppm (parts per million) refers to the resonance frequency of a reference substance whose chemical shift value is set equal to zero (Stordeur, 2007).

NMR-active nuclei in proteins are protons (¹H), nitrogen atoms (¹⁵N), and carbon atoms (¹³C).

However, the natural abundance of ¹⁵N and ¹³C nuclei is too low, so that they need to be artificially enriched in the protein of interest. Thus, the recombinant protein production is essential. As described by Stordeur, 2007, one problem in solution-state NMR recordings is the signal of protons from water. As they are represented in a higher concentration than the proteins NMR-active nuclei, their signals mask signals of the protein. The proton signal of water can be suppressed with the help of certain pulse sequences. However, proton signals that originate from the protein but lie in the resonance range of the water are also suppressed here and thus cannot be detected in the spectrum. Another possibility to avoid the presence of signals from water is to record spectra in proton-free solvents like deuterium oxide (D2O). Deuterium is a quadrupole nucleus and shows a far more reduced NMR-sensitivity than ¹H. Consequently, the water signal should be suppressed. However, amide protons of the protein are in permanent exchange with deuterium, which causes again an increase of the water signal in the spectrum while at the same time reducing the signals of the amides. Only inert amides, like those in a hydrogen-bond network, are shielded. In summary, complete data sets can only be obtained when spectra in both solvents, water and deuterium oxide, are recorded (Stordeur, 2007).

Normally, the first heteronuclear two-dimensional (2D) spectrum recorded for the assignment of resonances in protein NMR is the heteronuclear single quantum coherence (HSQC) spectrum (or the heteronuclear multiple quantum coherence (HMQC) spectrum with increased sensitivity but lower dispersion). Every observed peak can be assigned to a particular residue of the protein. Furthermore, indications for folded protein species can be gained by analyzing the dispersion and distribution of recorded peaks. Well-dispersed spectra without any clustering of signals point towards folded protein species under investigation. The HSQC experiment is based on two insensitive nuclei enhanced by polarization transfer (INEPT) pulses, which transfer the magnetization of the protons to the directly-attached heteronucleus (¹⁵N or ¹³C) and back to the proton where signals evolve.

The HSQC experiment is sensitive and can be recorded in a relatively short time. Thus, it

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makes it an ideal tool for initial screening purposes concerning sample stability and folding issues. Additionally, the experiment can be used for screening of binding interfaces when inhibitors or other binding partners are present. Here, spectra of holo- and apo-state of the protein are compared concerning their chemical shift properties. Furthermore, this kind of experiment can be used for the analysis of molecular dynamics in proteins by relaxation studies. In sum, HSQC experiments are particularly suited to investigate the mechanism behind proton channeling in VSDs.

Up to now, there is only one solution-state NMR data set for the hHV1 channel available (Letts, 2014). J. A. Letts worked with a shortened, 138 aa version of hHV1 with a truncated N- and C-terminus. He recorded [¹⁵N,¹H]-HSQC spectra of uniformly ²H,¹⁵N-labeled ∆N∆ChHV1 and assigned 82 % of the residues by ¹⁵N-selective labeling of the protein with 12 from 20 aa.

Interestingly, paramagnetic relaxation enhancement (PRE) measurements, used for distance restraint determination, revealed an unfolded protein structure in LPPG micelles although the initial HSQC-peak distribution looked quite promising. This means, NMR is subject to a constant back and forth between sample and spectra quality that is not necessarily compliant.

Intensive screening procedures are necessary. Expression host systems, purification strategies, detergent properties and many more parameters have to be adjusted to obtain folded protein species with well-dispersed peaks in NMR spectra recordings. However, it could be demonstrated that studying the VSDs by solution-state NMR is possible.

To sum up, the description and analysis of mechanistic features of channeling processes has to be based on a variety of different techniques to explain nature as close as possible. For all the above-mentioned experimental approaches, cell-free protein synthesis offers numerous advantages over the conventional in vivo expression systems, which includes an easier handling of protein labeling (e.g. with heavy isotopes), ligand/inhibitor additions promoting protein folding and stability, scrambling inhibitor suppressions and, especially for membrane proteins, the direct addition and easy screening of membrane mimetics. This makes it an ideal tool to study voltage-gated proton channels. The cell-free synthesis of this protein class is not described in the literature so far. Nevertheless, the synthesis of other voltage-gated channels could be successfully shown. Obtained membrane protein batches were correctly folded, stable, and functional (Deniaud et al., 2010; Kovácsová et al., 2015; Renauld et al., 2017).

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