Ümit Akbey

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]

Optimum levels of exchangeable protons in perdeuterated proteins for proton detection in MAS solid-state NMR spectroscopy.

We present a systematic study of the effect of the level of exchangeable protons on the observed amide proton linewidth obtained in perdeuterated proteins. Decreasing the amount of D2O employed in the crystallization buffer from 90 to 0%, we observe a fourfold increase in linewidth for both 1H and 15N resonances. At the same time, we find a gradual increase in the signal-to-noise ratio (SNR) for 1H–15N correlations in dipolar coupling based experiments for H2O concentrations of up to 40%. Beyond 40%, a significant reduction in SNR is observed. Scalar-coupling based 1H–15N correlation experiments yield a nearly constant SNR for samples prepared with ≤30% H2O. Samples in which more H2O is employed for crystallization show a significantly reduced NMR intensity. Calculation of the SNR by taking into account the reduction in 1H T1 in samples containing more protons (SNR per unit time), yields a maximum SNR for samples crystallized using 30 and 40% H2O for scalar and dipolar coupling based experiments, respectively. A sensitivity gain of 3.8 is obtained by increasing the H2O concentration from 10 to 40% in the CP based experiment, whereas the linewidth only becomes 1.5 times broader. In general, we find that CP is more favorable compared to INEPT based transfer when the number of possible 1H,1H interactions increases. At low levels of deuteration (≥60% H2O in the crystallization buffer), resonances from rigid residues are broadened beyond detection. All experiments are carried out at MAS frequency of 24xa0kHz employing perdeuterated samples of the chicken α-spectrin SH3 domain.

Neurotoxin II Bound to Acetylcholine Receptors in Native Membranes Studied by Dynamic Nuclear Polarization NMR

Methods enabling structural studies of membrane-integrated receptor systems without the necessity of purification provide an attractive perspective in membrane protein structural and molecular biology. This has become feasible in principle since the advent of dynamic nuclear polarization (DNP) magic-angle-spinning NMR spectroscopy, which delivers the required sensitivity. In this pilot study, we observed well-resolved solid-state NMR spectra of extensively (13)C-labeled neurotoxin II bound to the nicotinic acetylcholine receptor (nAChR) in native membranes. We show that TOTAPOL, a biradical required for DNP, is localized at membrane and protein surfaces. The concentration of active, membrane-attached biradical decreases with time, probably because of reactive components of the membrane preparation. An optimal distribution of active biradical has strong effects on the NMR data. The presence of inactive TOTAPOL in membrane-proximal situations but active biradical in the surrounding water/glycerol glass leads to well-resolved spectra, yet a considerable enhancement (ε = 12) is observed. The resulting spectra of a protein ligand bound to its receptor are paving the way for further DNP investigations of proteins embedded in native membrane patches.

Fast passage dynamic nuclear polarization on rotating solids

Magic Angle Spinning (MAS) Dynamic Nuclear Polarization (DNP) has proven to be a very powerful way to improve the signal to noise ratio of NMR experiments on solids. The experiments have in general been interpreted considering the Solid-Effect (SE) and Cross-Effect (CE) DNP mechanisms while ignoring the influence of sample spinning. In this paper, we show experimental data of MAS-DNP enhancements of (1)H and (13)C in proline and SH3 protein in glass forming water/glycerol solvent containing TOTAPOL. We also introduce a theoretical model that aims at explaining how the nuclear polarization is built in MAS-DNP experiments. By using Liouville space based simulations to include relaxation on two simple spin models, {electron-nucleus} and {electron-electron-nucleus}, we explain how the basic MAS-SE-DNP and MAS-CE-DNP processes work. The importance of fast energy passages and short level anti-crossing is emphasized and the differences between static DNP and MAS-DNP is explained. During a single rotor cycle the enhancement in the {electron-electron-nucleus} system arises from MAS-CE-DNP involving at least three kinds of two-level fast passages: an electron-electron dipolar anti-crossing, a single quantum electron MW encounter and an anti-crossing at the CE condition inducing nuclear polarization in- or decrements. Numerical, powder-averaged, simulations were performed in order to check the influence of the experimental parameters on the enhancement efficiencies. In particular we show that the spinning frequency dependence of the theoretical MAS-CE-DNP enhancement compares favorably with the experimental (1)H and (13)C MAS-DNP enhancements of proline and SH3.

Cryogenic temperature effects and resolution upon slow cooling of protein preparations in solid state NMR

X-ray crystallography using synchrotron radiation and the technique of dynamic nuclear polarization (DNP) in nuclear magnetic resonance (NMR) require samples to be kept at temperatures below 100xa0K. Protein dynamics are poorly understood below the freezing point of water and down to liquid nitrogen temperatures. Therefore, we investigate the α-spectrin SH3 domain by magic angle spinning (MAS) solid state NMR (ssNMR) at various temperatures while cooling slowly. Cooling down to 95xa0K, the NMR-signals of SH3 first broaden and at lower temperatures they separate into several peaks. The coalescence temperature differs depending on the individual residue. The broadening is shown to be inhomogeneous by hole-burning experiments. The coalescence behavior of 26 resolved signals (of 62) was compared to water proximity and crystal structure Debye–Waller factors (B-factors). Close proximity to the solvent and large B-factors (i.e. mobility) lead, generally, to a higher coalescence temperature. We interpret a high coalescence temperature as indicative of a large number of magnetically inequivalent populations at cryogenic temperature.

Self-Assembly of Dendronized Perylene Bisimides into Complex Helical Columns

The synthesis of perylene 3,4:9,10-tetracarboxylic acid bisimides (PBIs) dendronized with first-generation dendrons containing 0 to 4 methylenic units (m) between the imide group and the dendron, (3,4,5)12G1-m-PBI, is reported. Structural analysis of their self-organized arrays by DSC, X-ray diffraction, molecular modeling, and solid-state (1)H NMR was carried out on oriented samples with heating and cooling rates of 20 to 0.2 °C/min. At high temperature, (3,4,5)12G1-m-PBI self-assemble into 2D-hexagonal columnar phases with intracolumnar order. At low temperature, they form orthorhombic (m = 0, 2, 3, 4) and monoclinic (m = 1) columnar arrays with 3D periodicity. The orthorhombic phase has symmetry close to hexagonal. For m = 0, 2, 3, 4 ,they consist of tetramers as basic units. The tetramers contain a pair of two molecules arranged side by side and another pair in the next stratum of the column, turned upside-down and rotated around the column axis at different angles for different m. In contrast, for m = 1, there is only one molecule in each stratum, with a four-strata 2(1) helical repeat. All molecules face up in one column, and down in the second column, of the monoclinic cell. This allows close and extended π-stacking, unlike in the disruptive up-down alteration from the case of m = 0, 2, 3, 4. Most of the 3D structures were observed only by cooling at rates of 1 °C/min or less. This complex helical self-assembly is representative for other classes of dendronized PBIs investigated for organic electronics and solar cells.

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.

1H Solid-State NMR Investigation of Structure and Dynamics of Anhydrous Proton Conducting Triazole-Functionalized Siloxane Polymers

(1)H MAS solid-state NMR methods are applied to elucidate the conduction mechanism of an anhydrous proton conducting triazole-functionalized polysiloxane. At temperatures below T = 260 K, hydrogen bonding between neighboring heterocycles is observed and a dimer formation can be excluded. From the temperature dependence of (1)H MAS NMR spectra, different dynamic processes of the triazole ring contributing to the proton conduction process are qualitatively and quantitatively analyzed and detailed insight into the conduction mechanism and temperature-dependent structural changes is obtained. Although the dynamics processes on the molecular level are qualitatively in good agreement with the findings from macroscopic conductivity measurements, temperature-dependent factors on mesoscopic scales beyond the local molecular mobility influence the macroscopic conductivity and hamper quantitative interpretation.

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.

Triple Resonance Cross‐Polarization for More Sensitive 13C MAS NMR Spectroscopy of Deuterated Proteins

Save the last WALTZ for me: the use of simultaneous proton and deuterium cross-polarization for (13)C CPMAS NMR spectroscopy in highly deuterated proteins is discussed. The aim of the new method introduced herein, triple-resonance cross-polarization, is to increase the sensitivity of the carbon-detected methods in such systems.