Solid-State NMR Spectroscopy for Studying Microtubules and Microtubule-Associated Proteins.

In this chapter, we describe the preparatory and spectroscopic procedures for conducting solid-state NMR experiments on microtubules (MTs) obtained from human cells and their complexes with microtubule-associated proteins (MAPs). Next to labeling and functional assembly of MTs and MT-MAP complexes, we discuss solid-state NMR approaches, including fast MAS and hyperpolarization methods that can be used to examine these systems. Such studies can provide novel insight into the dynamic properties of MTs and MT-MAP complexes.

Mode of action of teixobactins in cellular membranes.

The natural antibiotic teixobactin kills pathogenic bacteria without detectable resistance. The difficult synthesis and unfavourable solubility of teixobactin require modifications, yet insufficient knowledge on its binding mode impedes the hunt for superior analogues. Thus far, teixobactins are assumed to kill bacteria by binding to cognate cell wall precursors (Lipid II and III). Here we present the binding mode of teixobactins in cellular membranes using solid-state NMR, microscopy, and affinity assays. We solve the structure of the complex formed by an improved teixobactin-analogue and Lipid II and reveal how teixobactins recognize a broad spectrum of targets. Unexpectedly, we find that teixobactins only weakly bind to Lipid II in cellular membranes, implying the direct interaction with cell wall precursors is not the sole killing mechanism. Our data suggest an additional mechanism affords the excellent activity of teixobactins, which can block the cell wall biosynthesis by capturing precursors in massive clusters on membranes.

High-resolution NMR studies of antibiotics in cellular membranes.

The alarming rise of antimicrobial resistance requires antibiotics with unexploited mechanisms. Ideal templates could be antibiotics that target the peptidoglycan precursor lipid II, known as the bacterial Achilles heel, at an irreplaceable pyrophosphate group. Such antibiotics would kill multidrug-resistant pathogens at nanomolecular concentrations without causing antimicrobial resistance. However, due to the challenge of studying small membrane-embedded drug-receptor complexes in native conditions, the structural correlates of the pharmaceutically relevant binding modes are unknown. Here, using advanced highly sensitive solid-state NMR setups, we present a high-resolution approach to study lipid II-binding antibiotics directly in cell membranes. On the example of nisin, the preeminent lantibiotic, we show that the native antibiotic-binding mode strongly differs from previously published structures, and we demonstrate that functional hotspots correspond to plastic drug domains that are critical for the cellular adaptability of nisin. Thereby, our approach provides a foundation for an improved understanding of powerful antibiotics.

Isolating Clusters of Light Elements in Molecular Sieves with Atom Probe Tomography.

Understanding the 3-D distribution and nature of active sites in heterogeneous catalysts is critical to developing structure-function relationships. However, this is difficult to achieve in microporous materials as there is little relative z-contrast between active and inactive framework elements (e.g., Al, O, P, and Si), making them difficult to differentiate with electron microscopies. We have applied atom probe tomography (APT), currently the only nanometer-scale 3-D microscopy to offer routine light element contrast, to the methanol-to-hydrocarbons (MTH) catalyst SAPO-34, with Si as the active site, which may be present in the framework as either isolated Si species or clusters (islands) of Si atoms. 29Si solid-state NMR data on isotopically enriched and natural abundance materials are consistent with the presence of Si islands, and the APT results have been complemented with simulations to show the smallest detectable cluster size as a function of instrument spatial resolution and detector efficiency. We have identified significant Si-Si affinity in the materials, as well as clustering of coke deposited by the MTH reaction (13CH3OH used) and an affinity between Brønsted acid sites and coke. A comparison with simulations shows that the ultimate spatial resolution that can be attained by APT applied to molecular sieves is 0.5-1 nm. Finally, the observed 13C clusters are consistent with hydrocarbon pool mechanism intermediates that are preferentially located in regions of increased Brønsted acidity.

Bridging the Gap between the Direct and Hydrocarbon Pool Mechanisms of the Methanol-to-Hydrocarbons Process.

After a prolonged effort over many years, the route for the formation of a direct carbon-carbon (C-C) bond during the methanol-to-hydrocarbon (MTH) process has very recently been unveiled. However, the relevance of the "direct mechanism"-derived molecules (that is, methyl acetate) during MTH, and subsequent transformation routes to the conventional hydrocarbon pool (HCP) species, are yet to be established. This important piece of the MTH chemistry puzzle is not only essential from a fundamental perspective, but is also important to maximize catalytic performance. The MTH process was probed over a commercially relevant H-SAPO-34 catalyst, using a combination of advanced solid-state NMR spectroscopy and operando UV/Vis diffuse reflectance spectroscopy coupled to an on-line mass spectrometer. Spectroscopic evidence is provided for the formation of (olefinic and aromatic) HCP species, which are indeed derived exclusively from the direct C-C bond-containing acetyl group of methyl acetate. New mechanistic insights have been obtained from the MTH process, including the identification of hydrocarbon-based co-catalytic organic reaction centers.

Proton and Carbon-13 Dynamic Nuclear Polarization of Methylated ß-Cyclodextrins.

1H and 13C dynamic nuclear polarizations have been studied in 13C-enriched ß-cyclodextrins doped with (2,2,6,6-tetramethylpiperidin-1-yl)oxyl free radical. 1H and 13C polarizations raised above 7.5 and 7%, respectively, and for both nuclear species, the transfer of polarization from the electron spins appears to be consistent with a thermal mixing scenario for a concentration of 9 13C nuclei per molecule. When the concentration is increased to 21 13C nuclei per molecule, a decrease in the spin-lattice relaxation and polarization buildup rates is observed. This reduction is associated with the bottleneck effect induced by the decrease in the number of electron spins per nucleus when both the nuclear spin-lattice relaxation and the polarization occur through the electron non-Zeeman reservoir. 13C nuclear spin-lattice relaxation has been studied in the 1.8-340 K range, and the effects of internal molecular motions and of the free radicals on the relaxation are discussed. 13C hyperpolarization performances and room-temperature spin-lattice relaxation times show that these are promising materials for future biomedical applications.