Distance tuneable integral membrane protein containing floating bilayers via in situ directed self-assembly.

Model membranes allow for structural and biophysical studies on membrane biochemistry at the molecular level, albeit on systems of reduced complexity which can limit biological accuracy. Floating supported bilayers offer a means of producing planar lipid membrane models not adhered to a surface, which allows for improved accuracy compared to other model membranes. Here we communicate the incorporation of an integral membrane protein complex, the multidomain ß-barrel assembly machinery (Bam), into our recently developed in situ self-assembled floating supported bilayers. Using neutron reflectometry and quartz crystal microbalance measurements we show this sample system can be fabricated using a two-step self-assembly process. We then demonstrate the complexity of the model membrane and tuneability of the membrane-to-surface distance using changes in the salt concentration of the bulk solution. Results demonstrate an easily fabricated, biologically accurate and tuneable membrane assay system which can be utilized for studies on integral membrane proteins within their native lipid matrix.

High-resolution structure of the alcohol dehydrogenase domain of the bifunctional bacterial enzyme AdhE.

The bifunctional alcohol/aldehyde dehydrogenase (AdhE) comprises both an N-terminal aldehyde dehydrogenase (AldDH) and a C-terminal alcohol dehydrogenase (ADH). In vivo, full-length AdhE oligomerizes into long oligomers known as spirosomes. However, structural analysis of AdhE is challenging owing to the heterogeneity of the spirosomes. Therefore, the domains of AdhE are best characterized separately. Here, the structure of ADH from the pathogenic Escherichia coli O157:H7 was determined to 1.65 Šresolution. The dimeric crystal structure was confirmed in solution by small-angle X-ray scattering.

ß-Lactamases and ß-Lactamase Inhibitors in the 21st Century.

The ß-lactams retain a central place in the antibacterial armamentarium. In Gram-negative bacteria, ß-lactamase enzymes that hydrolyze the amide bond of the four-membered ß-lactam ring are the primary resistance mechanism, with multiple enzymes disseminating on mobile genetic elements across opportunistic pathogens such as Enterobacteriaceae (e.g., Escherichia coli) and non-fermenting organisms (e.g., Pseudomonas aeruginosa). ß-Lactamases divide into four classes; the active-site serine ß-lactamases (classes A, C and D) and the zinc-dependent or metallo-ß-lactamases (MBLs; class B). Here we review recent advances in mechanistic understanding of each class, focusing upon how growing numbers of crystal structures, in particular for ß-lactam complexes, and methods such as neutron diffraction and molecular simulations, have improved understanding of the biochemistry of ß-lactam breakdown. A second focus is ß-lactamase interactions with carbapenems, as carbapenem-resistant bacteria are of grave clinical concern and carbapenem-hydrolyzing enzymes such as KPC (class A) NDM (class B) and OXA-48 (class D) are proliferating worldwide. An overview is provided of the changing landscape of ß-lactamase inhibitors, exemplified by the introduction to the clinic of combinations of ß-lactams with diazabicyclooctanone and cyclic boronate serine ß-lactamase inhibitors, and of progress and strategies toward clinically useful MBL inhibitors. Despite the long history of ß-lactamase research, we contend that issues including continuing unresolved questions around mechanism; opportunities afforded by new technologies such as serial femtosecond crystallography; the need for new inhibitors, particularly for MBLs; the likely impact of new ß-lactam:inhibitor combinations and the continuing clinical importance of ß-lactams mean that this remains a rewarding research area.

An acid-compatible co-polymer for the solubilization of membranes and proteins into lipid bilayer-containing nanoparticles.

The fundamental importance of membrane proteins in drug discovery has meant that membrane mimetic systems for studying membrane proteins are of increasing interest. One such system has been the amphipathic, negatively charged poly(styrene-co-maleic acid) (SMA) polymer to form "SMA Lipid Particles" (SMALPs) which have been widely adopted to solubilize membrane proteins directly from the cell membrane. However, SMALPs are only soluble under basic conditions and precipitate in the presence of divalent cations required for many downstream applications. Here, we show that the positively charged poly(styrene-co-maleimide) (SMI) forms similar nanoparticles with comparable efficiency to SMA, whilst remaining functional at acidic pH and compatible with high concentrations of divalent cations. We have performed a detailed characterization of the performance of SMI that enables a direct comparison with similar data published for SMA. We also demonstrate that SMI is capable of extracting proteins directly from the cell membrane and can solubilize functional human G-protein coupled receptors (GPCRs) expressed in cultured HEK 293T cells. "SMILPs" thus provide an alternative membrane solubilization method that successfully overcomes some of the limitations of the SMALP method.

UK and European Union public and charitable funding from 2008 to 2013 for bacteriology and antibiotic research in the UK: an observational study.

BACKGROUND: Since the 1990s, the number of new antibacterial drugs has plummeted and the number of antibiotic-resistant infections has risen, which has decreased the effective treatment of many disorders, including sepsis. We aimed to assess whether funding for bacteriology and antibiotic research to UK researchers had increased in response to this global crisis. METHODS: We systematically searched websites and databases of agencies that fund research in the UK to identify publicly and charitably funded projects from financial years 2008 to 2013 within the specialties of bacteriology and antibiotic research. We created a database to identify the projects funded. Grants awarded in euros were converted to pounds sterling (€1=£0·86). FINDINGS: We identified 609 projects within the specialty of bacteriology, 196 (32·2%) of which were on antibiotics. Of £13 846·1 million of available research funding, £269·2 million (1·9%) was awarded to bacteriology projects and £95·0 million (0·7%) was awarded for research on antibiotics. Additionally, £181·4 million in European Union (EU) funding was awarded to antibiotic research consortia including researchers based within the UK, including two EU Innovative Medicines Initiative awards, totalling £85·2 million. INTERPRETATION: To increase awareness of who funds antibiotic research and to facilitate priority setting and funding decisions, funding organisations need to be aware of the breadth and depth of present funding as a baseline by which funding from 2014 onwards can be measured and so that informed decisions about the future level of funding can be made. To resolve the crisis of antibiotic resistance, present levels of funding are inadequate and should be increased substantially. FUNDING: British Society for Antimicrobial Chemotherapy.