Determining the Orientation of Porins in Planar Lipid Bilayers.

Single-channel planar lipid bilayer (PLB) recording of bacterial porins has revealed molecular details of transport across the outer membrane of Gram-negative bacteria, including antibiotic permeation and protein translocation. To explore directional transport processes across cellular membranes, the orientation of porins or other pore-forming proteins must be established in a lipid bilayer prior to experimentation. Here, we describe a direct method for determining the orientation of porins in a PLB-with a focus on E. coli OmpF-by using targeted covalent modification of cysteine mutants. Each of the two possible orientations can be correlated with the porin conductance asymmetry, such that thereafter an I-V curve taken at the start of an experiment will suffice to establish orientation.

Transmembrane Epitope Delivery by Passive Protein Threading through the Pores of the OmpF Porin Trimer.

Trimeric porins in the outer membrane (OM) of Gram-negative bacteria are the conduits by which nutrients and antibiotics diffuse passively into cells. The narrow gateways that porins form in the OM are also exploited by bacteriocins to translocate into cells by a poorly understood process. Here, using single-channel electrical recording in planar lipid bilayers in conjunction with protein engineering, we explicate the mechanism by which the intrinsically unstructured N-terminal translocation domain (IUTD) of the endonuclease bacteriocin ColE9 is imported passively across the Escherichia coli OM through OmpF. We show that the import is dominated by weak interactions of OmpF pores with binding epitopes within the IUTD that are orientationally biased and result in the threading of over 60 amino acids through 2 subunits of OmpF. Single-molecule kinetic analysis demonstrates that the IUTD enters from the extracellular side of OmpF and translocates to the periplasm where the polypeptide chain does an about turn in order to enter a neighboring subunit, only for some of these molecules to pop out of this second subunit before finally re-entering to form a stable complex. These intimately linked transport/binding processes generate an essentially irreversible, hook-like assembly that constrains an import activating peptide epitope between two subunits of the OmpF trimer.

Catalytic site-selective substrate processing within a tubular nanoreactor.

Chemists have long sought the ability to modify molecules precisely when presented with several sites of similar reactivity. We reasoned that the confinement of substrates within nanostructures might permit site-selective reactions unachievable in bulk solution, even with sophisticated reagents. In particular, the stretching and alignment of polymers within nanotubes might allow site-specific cleavage or modification. To explore this proposition, macromolecular disulfide substrates were elongated within members of a collection of tubular protein nanoreactors, which contained cysteine residues positioned at different locations along the length of each tube. For each nanoreactor, we defined the reactive location by using a set of polymer substrates (site-selectivity) and which of the two sulfur atoms was attacked (regioselectivity), and found that disulfide interchange occurs with atomic precision. Our strategy has potential for the selective processing of a wide variety of biomacromolecules, and the chemistry and substrates might be generalized yet further by using alternative nanotubes.

Directional control of a processive molecular hopper.

Intrigued by the potential of nanoscale machines, scientists have long attempted to control molecular motion. We monitored the individual 0.7-nanometer steps of a single molecular hopper as it moved in an electric field along a track in a nanopore controlled by a chemical ratchet. The hopper demonstrated characteristics desired in a moving molecule: defined start and end points, processivity, no chemical fuel requirement, directional motion, and external control. The hopper was readily functionalized to carry cargos. For example, a DNA molecule could be ratcheted along the track in either direction, a prerequisite for nanopore sequencing.

A new class of hybrid secretion system is employed in Pseudomonas amyloid biogenesis.

Gram-negative bacteria possess specialised biogenesis machineries that facilitate the export of amyloid subunits for construction of a biofilm matrix. The secretion of bacterial functional amyloid requires a bespoke outer-membrane protein channel through which unfolded amyloid substrates are translocated. Here, we combine X-ray crystallography, native mass spectrometry, single-channel electrical recording, molecular simulations and circular dichroism measurements to provide high-resolution structural insight into the functional amyloid transporter from Pseudomonas, FapF. FapF forms a trimer of gated ß-barrel channels in which opening is regulated by a helical plug connected to an extended coil-coiled platform spanning the bacterial periplasm. Although FapF represents a unique type of secretion system, it shares mechanistic features with a diverse range of peptide translocation systems. Our findings highlight alternative strategies for handling and export of amyloid protein sequences.Gram-negative bacteria assemble biofilms from amyloid fibres, which translocate across the outer membrane as unfolded amyloid precursors through a secretion system. Here, the authors characterise the structural details of the amyloid transporter FapF in Pseudomonas.

Orientation of the OmpF Porin in Planar Lipid Bilayers.

The outer-membrane protein OmpF is an abundant trimeric general diffusion porin that plays a central role in the transport of antibiotics and colicins across the outer membrane of E. coli. Individual OmpF trimers in planar lipid bilayers (PLBs) show one of two current-voltage asymmetries, thus implying that insertion occurs with either the periplasmic or the extracellular end first. A method for establishing the orientation of OmpF in PLB was developed, based on targeted covalent modification with membrane-impermeant reagents of peripheral cysteine residues introduced near the periplasmic or the extracellular entrance. By correlating the results of the modification experiments with measurements of current asymmetry or the sidedness of binding of the antibiotic enrofloxacin, OmpF orientation could be quickly determined in subsequent experiments under a variety of conditions. Our work will allow the precise interpretation of past and future studies of antibiotic permeation and protein translocation through OmpF and related porins.

Characterizing the dynamics of the leader-linker interaction in the glycine riboswitch with site-directed spin labeling.

Site-directed spin labeling with continuous wave electron paramagnetic resonance (EPR) spectroscopy was utilized to characterize dynamic features of the kink-turn motif formed through a leader-linker interaction in the Vibrio cholerae glycine riboswitch. Efficient incorporation of spin-labels into select sites within the phosphate backbone of the leader-linker region proceeded via splinted ligation of chemically synthesized spin-labeled oligonucleotides to in vitro transcribed larger RNA fragments. The resultant nitroxide EPR line shapes have spectral characteristics consistent with a kink-turn motif and reveal differential backbone dynamics that are modulated by the presence of magnesium, potassium, and glycine.