Membrane proteins are known to exert many essential biological functions by forming complexes in cell membranes. An example refers to the β-barrel assembly machinery (BAM), a 200 kDa pentameric complex containing BAM proteins A–E that catalyzes the essential process of protein insertion into the outer membrane of gram-negative bacteria. While progress has been made in capturing three-dimensional structural snapshots of the BAM complex, the role of the lipoprotein BamC in the complex assembly in functional lipid bilayers has remained unclear. We have devised a component-selective preparation scheme to directly study BamC as part of the entire BAM complex in lipid bilayers. Combination with proton-detected solid-state NMR methods allowed us to probe the structure, dynamics, and supramolecular topology of full-length BamC embedded in the entire complex in lipid bilayers. Our approach may help decipher how individual proteins contribute to the dynamic formation and functioning of membrane protein complexes in membranes.

For a long time, solid-state nuclear magnetic resonance (ssNMR) has been employed to study complex biomolecular systems at the detailed chemical, structural, or dynamic level. Recent progress in high-resolution and high-sensitivity ssNMR, in combination with innovative sample preparation and labeling schemes, offers novel opportunities to study proteins in their native setting irrespective of the molecular tumbling rate. This protocol describes biochemical preparation schemes to obtain cellular samples of both soluble as well as insoluble or membrane-associated proteins in bacteria. To this end, the protocol is suitable for studying a protein of interest in both whole cells and in cell envelope or isolated membrane preparations. In the first stage of the procedure, an appropriate strain of Escherichia coli (DE3) is transformed with a plasmid of interest harboring the protein of interest under the control of an inducible T7 promoter. Next, the cells are adapted to grow in minimal (M9) medium. Before the growth enters stationary phase, protein expression is induced, and shortly thereafter, the native E. coli RNA polymerase is inhibited using rifampicin for targeted labeling of the protein of interest. The cells are harvested after expression and prepared for ssNMR rotor filling. In addition to conventional ¹³C/¹⁵N-detected ssNMR, we also outline how these preparations can be readily subjected to multidimensional ssNMR experiments using dynamic nuclear polarization (DNP) or proton (¹H) detection schemes. We estimate that the entire preparative procedure until NMR experiments can be started takes 3–5 days.

Although the local geometry of Mo in Mo/HZSM-5 has been characterized before, we present a systematic way to manipulate the configuration of Mo and link it to its catalytic properties. The location and geometry of cationic Mo-complexes, the precursor of the active metal site for methane dehydroaromatization, are altered by directing the way they anchor to the framework of the zeolite. The feature used to direct the anchoring of Mo is the location of Al in the zeolite framework. According to DFT calculations, the local geometry of Mo should change, while UV-vis and pyridine FTIR spectroscopy indicated differences in the dispersion of Mo. Both aspects, however, did not influence the catalytic behavior of Mo/HZSM-5, indicating that as long as enough isolated Mo species are present inside the pores of the zeolite, the catalytic behavior is unaffected. This paves the way to better understand how the Mo oxo precursor transforms into the active phase under the reaction conditions.

Incorporation of Ca in ZSM-5 results in a twofold increase of propylene selectivity (53 %), a total light-olefin selectivity of 90 %, and a nine times longer catalyst lifetime (throughput 792 g_MeOH g_catalyst ⁻¹) in the methanol-to-olefins (MTO) reaction. Analysis of the product distribution and theoretical calculations reveal that post-synthetic modification with Ca²⁺ leads to the formation of CaOCaOH⁺ that strongly weaken the acid strength of the zeolite. As a result, the rate of hydride transfer and oligomerization reactions on these sites is greatly reduced, resulting in the suppression of the aromatic cycle. Our results further highlight the importance of acid strength on product selectivity and zeolite lifetime in MTO chemistry.

The front cover artwork for Issue 19/2016 is provided by researchers from the Catalysis Engineering group at TU Delft (The Netherlands). The image shows that after impregnation with Ca, ZSM-5 becomes a “super-catalyst”, which is able to suppress formation of aromatics thus increasing selectivity to the desired propylene in MTO reaction. See the Full Paper itself at http://dx.doi.org/10.1002/cctc.201600650.