Charge neutralization of the active site glutamates does not limit substrate binding and transport by small multidrug resistance transporter EmrE.

EmrE, a small multidrug resistance transporter from Escherichia coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of two protons:one cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14 and E14 (one from each subunit in the antiparallel homodimer), in the primary binding site.

Structure and dynamics of the drug-bound bacterial transporter EmrE in lipid bilayers.

The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using F and H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F-TPP).

pH-Triggered Release from Polyamide Microcapsules Prepared by Interfacial Polymerization of a Simple Diester Monomer.

The majority of current pH-triggered release systems is designed to respond to either low or high pH. Encapsulants based on polyampholytes are an example of materials that can respond to both acidic and basic pH. However, polyampholyte-based encapsulants generally possess a low loading capacity and have difficulty retaining their small-molecule cargo.

The membrane localization domains of two distinct bacterial toxins form a 4-helix-bundle in solution.

Membrane localization domain (MLD) was first proposed for a 4-helix-bundle motif in the crystal structure of the C1 domain of Pasteurella multocida toxin (PMT). This structure motif is also found in the crystal structures of several clostridial glycosylating toxins (TcdA, TcdB, TcsL, and TcnA). The Ras/Rap1-specific endopeptidase (RRSP) module of the multifunctional autoprocessing repeats-in-toxins (MARTX) toxin produced by Vibrio vulnificus has sequence homology to the C1-C2 domains of PMT, including a putative MLD.

An efficient method and device for transfer of semisolid materials into solid-state NMR spectroscopy rotors.

The study of mass-limited biological samples by magic angle spinning (MAS) solid-state NMR spectroscopy critically relies upon the high-yield transfer of material from a biological preparation into the MAS rotor. This issue is particularly important for maintaining biological activity and hydration of semi-solid samples such as membrane proteins in lipid bilayers, pharmaceutical formulations, microcrystalline proteins and protein fibrils. Here we present protocols and designs for rotor-packing devices specifically suited for packing hydrated samples into Pencil-style 1.

Amphotericin forms an extramembranous and fungicidal sterol sponge.

For over 50 years, amphotericin has remained the powerful but highly toxic last line of defense in treating life-threatening fungal infections in humans with minimal development of microbial resistance. Understanding how this small molecule kills yeast is thus critical for guiding development of derivatives with an improved therapeutic index and other resistance-refractory antimicrobial agents. In the widely accepted ion channel model for its mechanism of cytocidal action, amphotericin forms aggregates inside lipid bilayers that permeabilize and kill cells.

Backbone and side-chain assignments of an effector membrane localization domain from Vibrio vulnificus MARTX toxin.

(1)H, (13)C, and (15)N chemical shift assignments are presented for the isolated four-helical bundle membrane localization domain from the domain of unknown function 5 (DUF5) effector (MLD(VvDUF5)) of the MARTX toxin from Vibrio vulnificus in its solution state. We have assigned 97% of all backbone and side-chain carbon atoms, including 96% of all backbone residues. Secondary chemical shift analysis using TALOS+ demonstrates four helices that align with those predicted by structure homology modeling using the MLDs of Pasteurella multocida toxin (PMT) and the clostridial TcdB and TcsL toxins as templates.

Backbone and side-chain resonance assignments of the membrane localization domain from Pasteurella multocida toxin.

(1)H, (13)C, and (15)N chemical shift assignments are presented for the isolated four-helical bundle membrane localization domain (MLD) from Pasteurella multocida toxin (PMT) in its solution state. We have assigned 99% of all backbone and side-chain carbon atoms, including 99% of all backbone residues excluding proline amide nitrogens. Secondary chemical shift analysis using TALOS+ demonstrates four helices, which align with those observed within the MLD in the crystal structure of the C-terminus of PMT (PDB 2EBF) and confirm the use of the available crystal structures as templates for the isolated MLDs.

Indirect detection of the 183W and 57Fe nuclei using 119Sn-relayed 1H,X correlation spectroscopy.

Recently reported triple-resonance Y-relayed (1)H,X correlation experiments have been utilized to characterize (183)W and (57)Fe chemical shifts using (119)Sn as the Y-relaying nucleus instead of the previously used (31)P. Application of an adaptation of Gudat's original INEPT/HMQC sequence results in a significant enhancement of the signal-to-noise (S/N) ratio for two-dimensional (119)Sn-relayed (1)H,(183)W and (1)H, (57)Fe correlation spectra with efficient detection of the transition metal nucleus in tungsten and iron complexes lacking an observable direct scalar coupling between the transition metal and any hydrogen nuclei. Strengths and shortcomings of the novel sequence and the original sequences reported by Gudat are discussed in the context of (119)Sn-relayed proton detection of very low frequency transition metal nuclei.