Eline J. Koers

Solid-State NMR Spectroscopy on Cellular Preparations Enhanced by Dynamic Nuclear Polarization†

Solid-state NMR (ssNMR) spectroscopy offers increasing possibilities to study complex biomolecules at the atomic level. An important target area concerns membrane-associated proteins, which can be investigated by ssNMR methods after reconstitution in synthetic bilayers. While such preparations allow examination of functional aspects of the protein of interest, the influence of the native cellular environment on protein structure and function cannot be monitored. Very recently, we introduced a general approach aimed at determining complex molecular structures, including integral membrane proteins, in their native cellular environment by ssNMR under magic-angle-spinning (MAS) conditions. Using dedicated sample-preparation routes, we demonstrated that high-resolution ssNMR spectra can be obtained on uniformly C,N-labeled preparations of Escherichia coli whole cells (WC) and cell envelopes (CE). Both CE and WC morphology are preserved under standard ssNMR experimental conditions and the corresponding C and N crosspolarization (CP-MAS) spectra are invariant over time. However, with increasing levels of molecular complexity, especially in the case of WC preparations, spectroscopic sensitivity becomes a critical factor. In recent years, dynamic nuclear polarization (DNP) has developed into a routine tool to increase the sensitivity of multidimensional ssNMR. DNP enhancements of up to 148fold have been obtained on micro/nanocrystalline biomolecular samples, including an amyloidogenic peptide and a deuterated protein, 6] while enhancements between 18and 46fold have been reported for membrane-embedded polypeptides, purple membrane preparations, and bacteriophages. Here, we investigated the use of DNP to conduct ssNMR studies on C,N-labeled preparations of E. coli WC overproducing the integral outer membrane protein PagL. In Figure 1, we compared C and N CP-MAS spectra of uniformly C,N-labeled WC with the CE isolated from PagL-overproducing E. coli cells, recorded in the presence and absence of microwave irradiation. At higher temperatures (271 K), ssNMR spectra of the E. coli CE had previously revealed atomic details of PagL as well as endogenous membrane-associated macromolecules, including the major lipoprotein Lpp and non-proteinaceous components such as lipopolysaccharides (LPS), peptidoglycans (PG), and phospholipids. Under low-temperature (LT) DNP conditions, we observed significant DNP enhancement factors for both preparations in spectral regions characteristic for protein signals (aliphatic C resonances: d = 50–55 ppm, amide N backbone and side-chain resonances at about 120 and 80–30 ppm) as well as for C signals of endogenous

Platinum-Promoted Ga/Al2O3 as Highly Active, Selective, and Stable Catalyst for the Dehydrogenation of Propane†

A novel catalyst material for the selective dehydrogenation of propane is presented. The catalyst consists of 1000 ppm Pt, 3 wt % Ga, and 0.25 wt % K supported on alumina. We observed a synergy between Ga and Pt, resulting in a highly active and stable catalyst. Additionally, we propose a bifunctional active phase, in which coordinately unsaturated Ga3+ species are the active species and where Pt functions as a promoter.

Structural characterization of 13C-enriched humins and alkali-treated 13C humins by 2D solid-state NMR

Humin by-products are formed during the acid-catalyzed dehydration of carbohydrates to bio-based platform molecules, such as hydroxymethylfurfural and levulinic acid. The molecular structure of these humins has not yet been unequivocally established. 1D 13C solid-state NMR data reported have, for example, provided considerable insight, but do not allow for the unambiguous assignment of key structural motifs. Complementary (2D) techniques are needed to gain additional insight into the molecular structure of humins. Here, the preparation of 13C-enriched humins is reported, together with the reactive solubilization of these labeled humins and their characterization with complementary 1D and 2D solid-state NMR techniques. 1D cross polarization (CP) and direct excitation (DE) 13C solid-state NMR spectra, 2D 13C-detected double-quantum single-quantum (DQSQ) as well as 2D 1H-detected heteronuclear correlation (HETCOR) were recorded with different excitation schemes. These experiments unambiguously established that the original humins have a furan-rich structure with aliphatic linkers and allowed for a refinement of the molecular structure proposed previously. Solid-state NMR data of alkali-treated 13C-labeled humins showed that an arene-rich structure is formed at the expense of the furanic network during alkaline pretreatment.

Dynamic nuclear polarization NMR spectroscopy: revealing multiple conformations in lipid-anchored peptide vaccines.

Keywords: Alzheimers disease ; dynamic nuclear polarization ; liposomal vaccines ; NMR spectroscopy ; -amyloid peptide Reference EPFL-ARTICLE-190124doi:10.1002/anie.201303374View record in Web of Science Record created on 2013-11-04, modified on 2017-05-12

Biomolecular DNP‐Supported NMR Spectroscopy using Site‐Directed Spin Labeling

Dynamic nuclear polarization (DNP) has been shown to greatly enhance spectroscopic sensitivity, creating novel opportunities for NMR studies on complex and large molecular assemblies in life and material sciences. In such applications, however, site-specificity and spectroscopic resolution become critical factors that are usually difficult to control by current DNP-based approaches. We have examined in detail the effect of directly attaching mono- or biradicals to induce local paramagnetic relaxation effects and, at the same time, to produce sizable DNP enhancements. Using a membrane-embedded ion channel as an example, we varied the degree of paramagnetic labeling and the location of the DNP probes. Our results show that the creation of local spin clusters can generate sizable DNP enhancements while preserving the intrinsic benefits of paramagnetic relaxation enhancement (PRE)-based NMR approaches. DNP using chemical labeling may hence provide an attractive route to introduce molecular specificity into DNP studies in life science applications and beyond.

Insight into the Supramolecular Architecture of Intact Diatom Biosilica from DNP-Supported Solid-State NMR Spectroscopy

Diatom biosilica is an inorganic/organic hybrid with interesting properties. The molecular architecture of the organic material at the atomic and nanometer scale has so far remained unknown, in particular for intact biosilica. A DNP-supported ssNMR approach assisted by microscopy, MS, and MD simulations was applied to study the structural organization of intact biosilica. For the first time, the secondary structure elements of tightly biosilica-associated native proteins in diatom biosilica were characterized in situ. Our data suggest that these proteins are rich in a limited set of amino acids and adopt a mixture of random-coil and β-strand conformations. Furthermore, biosilica-associated long-chain polyamines and carbohydrates were characterized, thereby leading to a model for the supramolecular organization of intact biosilica.