Open Access

Membrane proteins can be examined in near-native lipid-bilayer environments with the advent of polymer-encapsulated nanodiscs. These nanodiscs self-assemble directly from cellular membranes, allowing in vitro probing of membrane proteins with techniques that have previously been restricted to soluble or detergent-solubilized proteins. Often, however, the high charge densities of existing polymers obstruct bioanalytical and preparative techniques. Thus, the authors aim to fabricate electroneutral—yet water-soluble—polymer nanodiscs. By attaching a sulfobetaine group to the commercial polymers DIBMA and SMA(2:1), these polyanionic polymers are converted to the electroneutral maleimide derivatives, Sulfo-DIBMA and Sulfo-SMA(2:1). Sulfo-DIBMA and Sulfo-SMA(2:1) readily extract proteins and phospholipids from artificial and cellular membranes to form nanodiscs. Crucially, the electroneutral nanodiscs avert unspecific interactions, thereby enabling new insights into protein–lipid interactions through lab-on-a-chip detection and in vitro translation of membrane proteins. Finally, the authors create a library comprising thousands of human membrane proteins and use proteome profiling by mass spectrometry to show that protein complexes are preserved in electroneutral nanodiscs.

Highlights • Transporter proteins are essential for cellular function, but their functions are poorly characterized due to the lack of direct assays. • New workflow for transporter functional characterization combines cell-free transporter protein expression and solid supported membrane-based electrophysiology. • The workflow can be executed in five days. • Five transporters from SMR, MFS, Nha, and MC families were functionally expressed and analyzed. • The assay can provide: substrate specificity, kinetic parameters, pH dependency, and mechanistic insights. Abstract Functional characterization of transporters is impeded by the high cost and technical challenges of current transporter assays. Thus, in this work, we developed a new characterization workflow that combines cell-free protein synthesis (CFPS) and solid supported membrane-based electrophysiology (SSME). For this, membrane protein synthesis was accomplished in a continuous exchange cell-free system (CECF) in the presence of nanodiscs. The resulting transporters expressed in nanodiscs were incorporated into proteoliposomes and assayed in the presence of different substrates using the surface electrogenic event reader. As a proof of concept, we validated this workflow to express and characterize five diverse transporters: the drug/H+-coupled antiporters EmrE and SugE, the lactose permease LacY, the Na+/H+ antiporter NhaA from Escherichia coli, and the mitochondrial carrier AAC2 from Saccharomyces cerevisiae. For all transporters kinetic parameters, such as KM, IMAX, and pH dependency, were evaluated. This robust and expedite workflow (e.g., can be executed within only five workdays) offers a convenient direct functional assessment of transporter protein activity and has the ability to facilitate applications of transporters in medical and biotechnological research.

PorA and PorH are two small membrane proteins from the outer membrane of Corynebacterium glutamicum, which have been shown to form heteromeric ion channels and to be post-translationally modified by mycolic acids. Any structural details of the channel could not be analyzed so far due to tremendous difficulties in the production of sufficient amounts of protein samples. Cell-free (CF) expression is a new and remarkably successful strategy for the production of membrane proteins for which toxicity, membrane targeting, and degradation are key issues. In addition, reaction conditions can easily be modified to modulate the quality of synthesized protein samples. We developed an efficient CF expression strategy to produce the channel subunits devoid of post-translational modifications. (15)N-labeled PorA and PorH samples were furthermore characterized by NMR and gave well resolved spectra, opening the way for structural studies. The comparison of ion channel activities of CF-expressed proteins with channels isolated from C. glutamicum gave clear insights on the influence of the mycolic acid modification of the two subunits on their functional properties.

Mycobacteria contain an outer membrane composed of mycolic acids and a large variety of other lipids. Its protective function is an essential virulence factor of Mycobacterium tuberculosis. Only OmpA, which has numerous homologs in Gram-negative bacteria, is known to form channels in the outer membrane of M. tuberculosis so far. Rv1698 was predicted to be an outer membrane protein of unknown function. Expression of rv1698 restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698 partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. These results indicated that Rv1698 provides an unspecific pore that can partially substitute for MspA. Lipid bilayer experiments demonstrated that purified Rv1698 is an integral membrane protein that indeed produces channels. The main single channel conductance is 4.5 +/- 0.3 nanosiemens in 1 M KCl. Zero current potential measurements revealed a weak preference for cations. Whole cell digestion of recombinant M. smegmatis with proteinase K showed that Rv1698 is surface-accessible. Taken together, these experiments demonstrated that Rv1698 is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Rv1698 has single homologs of unknown functions in Corynebacterineae and thus represents the first member of a new class of channel proteins specific for mycolic acid-containing outer membranes.

Immunosurveillance of tumor cells depends on NKp30, a major activating receptor of human natural killer (NK) cells. The human BCL2-associated athanogene 6 (BAG-6, also known as BAT3; 1126 amino acids) is a cellular ligand of NKp30. To date, little is known about the molecular details of this receptor ligand system. Within the current study, we have located the binding site of NKp30 to a sequence stretch of 250 amino acids in the C-terminal region of BAG-6 (BAG-6(686-936)). BAG-6(686-936) forms a noncovalent dimer of 57-59 kDa, which is sufficient for high affinity interaction with NKp30 (KD < 100 nM). As our most important finding, BAG-6(686-936) inhibits NKp30-dependent signaling, interferon-γ release, and degranulation of NK cells in the presence of malignantly transformed target cells. Based on these data, we show for the first time that BAG-6(686-936) comprises a subdomain of BAG-6, which is sufficient for receptor docking and inhibition of NKp30-dependent NK cell cytotoxicity as part of a tumor immune escape mechanism. These molecular insights provide an access point to restore tumor immunosurveillance by NK cells and to increase the efficacy of cellular therapies.