Sascha Lange

Dynamic nuclear polarization of deuterated proteins.

Magic-angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy has evolved as a robust and widely applicable technique for investigating the structure and dynamics of biological systems.[1–3] It is in fact rapidly becoming an indispensable tool in structural biology studies of amyloid,[4, 5] nanocrystalline,[6, 7] and membrane proteins.[8] However, it is clear that the low sensitivity of MAS experiments to directly detected 13C and 15N signals limits the utility of the approach, particularly when working with systems which are difficult to obtain in large quantities. This limit provides the impetus to develop methods to enhance the sensitivity of MAS experiments, the availability of which will undoubtedly broaden the applicability of the technique. Remarkable progress towards this goal has been achieved by incorporating high-frequency dynamic nuclear polarization (DNP) into the MAS NMR technique.[9–17] The DNP method exploits the microwave-driven transfer of polarization from a paramagnetic center, such as nitroxide free radical, to the nuclear spins, and has been demonstrated to produce uniformly polarized macromolecular samples. In principle signal enhancements, e = (γe/γI) ≈ 660 can be obtained for 1H and recently signal enhancements of e = 100–200 were observed in model compounds. However, in applications of DNP to MAS spectra of biological systems, including studies of lysozyme,[18] and bacteriorhodopsin,[16, 19, 20] the enhancements have been smaller, e = 40–50. An exception is the amyloidogenic peptide GNNQQNY7–13 which forms nanocrystals for which the proton T1 time is long and e ≈ 100.[21]

The effect of biradical concentration on the performance of DNP-MAS-NMR

With the technique of dynamic nuclear polarization (DNP) signal intensity in solid-state MAS-NMR experiments can be enhanced by 2-3 orders of magnitude. DNP relies on the transfer of electron spin polarization from unpaired electrons to nuclear spins. For this reason, stable organic biradicals such as TOTAPOL are commonly added to samples used in DNP experiments. We investigated the effects of biradical concentration on the relaxation, enhancement, and intensity of NMR signals, employing a series of samples with various TOTAPOL concentrations and uniformly (13)C, (15)N labeled proline. A considerable decrease of the NMR relaxation times (T(1), T(2)(∗), and T(1)(ρ)) is observed with increasing amounts of biradical due to paramagnetic relaxation enhancement (PRE). For nuclei in close proximity to the radical, decreasing T(1)(ρ) reduces cross-polarization efficiency and decreases in T(2)(∗) broaden the signal. Additionally, paramagnetic shifts of (1)H signals can cause further line broadening by impairing decoupling. On average, the combination of these paramagnetic effects (PE; relaxation enhancement, paramagnetic shifts) quenches NMR-signals from nuclei closer than 10Å to the biradical centers. On the other hand, shorter T(1) times allow the repetition rate of the experiment to be increased, which can partially compensate for intensity loss. Therefore, it is desirable to optimize the radical concentration to prevent additional line broadening and to maximize the signal-to-noise observed per unit time for the signals of interest.

Dynamic nuclear polarization enhanced NMR in the solid-state.

Nuclear magnetic resonance (NMR) spectroscopy is one of the most commonly used spectroscopic techniques to obtain information on the structure and dynamics of biological and chemical materials. A variety of samples can be studied including solutions, crystalline solids, powders and hydrated protein extracts. However, biological NMR spectroscopy is limited to concentrated samples, typically in the millimolar range, due to its intrinsic low sensitivity compared to other techniques such as fluorescence or electron paramagnetic resonance (EPR) spectroscopy.Dynamic nuclear polarization (DNP) is a method that increases the sensitivity of NMR by several orders of magnitude. It exploits a polarization transfer from unpaired electrons to neighboring nuclei which leads to an absolute increase of the signal-to-noise ratio (S/N). Consequently, biological samples with much lower concentrations can now be studied in hours or days compared to several weeks.This chapter will explain the different types of DNP enhanced NMR experiments, focusing primarily on solid-state magic angle spinning (MAS) DNP, its applications, and possible means of improvement.

Bacteriophage Tail-Tube Assembly Studied by Proton-Detected 4D Solid-State NMR

Abstract Obtaining unambiguous resonance assignments remains a major bottleneck in solid‐state NMR studies of protein structure and dynamics. Particularly for supramolecular assemblies with large subunits (>150 residues), the analysis of crowded spectral data presents a challenge, even if three‐dimensional (3D) spectra are used. Here, we present a proton‐detected 4D solid‐state NMR assignment procedure that is tailored for large assemblies. The key to recording 4D spectra with three indirect carbon or nitrogen dimensions with their inherently large chemical shift dispersion lies in the use of sparse non‐uniform sampling (as low as 2 %). As a proof of principle, we acquired 4D (H)COCANH, (H)CACONH, and (H)CBCANH spectra of the 20 kDa bacteriophage tail‐tube protein gp17.1 in a total time of two and a half weeks. These spectra were sufficient to obtain complete resonance assignments in a straightforward manner without use of previous solution NMR data.

Protein−Protein Interfaces Probed by Methyl Labeling and Proton-Detected Solid-State NMR Spectroscopy

Abstract Proton detection and fast magic‐angle spinning have advanced biological solid‐state NMR, allowing for the backbone assignment of complex protein assemblies with high sensitivity and resolution. However, so far no method has been proposed to detect intermolecular interfaces in these assemblies by proton detection. Herein, we introduce a concept based on methyl labeling that allows for the assignment of these moieties and for the study of protein−protein interfaces at atomic resolution.