Substrate Specificity within a Family of Outer Membrane Carboxylate Channels

Characterization of a large family of outer membrane channels from gram-negative bacteria suggest how they can thrive in nutrient-poor environments and how channel inactivation can contribute to antibiotic resistance

Partitioning of Individual Flexible Polymers into a Nanoscopic Protein Pore

Polymer dynamics are of fundamental importance in materials science, biotechnology, and medicine. However, very little is known about the kinetics of partitioning of flexible polymer molecules into pores of nanometer dimensions. We employed electrical recording to probe the partitioning of single poly(ethylene glycol) (PEG) molecules, at concentrations near the dilute regime, into the transmembrane β-barrel of individual protein pores formed from staphylococcal α-hemolysin (αHL). The interactions of the α-hemolysin pore with the PEGs (M(w) 940–6000 Da) fell into two classes: short-duration events (τ ∼ 20 μs), ∼85% of the total, and long-duration events (τ ∼ 100 μs), ∼15% of the total. The association rate constants (k(on)) for both classes of events were strongly dependent on polymer mass, and values of k(on) ranged over two orders of magnitude. By contrast, the dissociation rate constants (k(off)) exhibited a weak dependence on mass, suggesting that the polymer chains are largely compacted before they enter the pore, and do not decompact to a significant extent before they exit. The values of k(on) and k(off) were used to determine partition coefficients (Π) for the PEGs between the bulk aqueous phase and the pore lumen. The low values of Π are in keeping with a negligible interaction between the PEG chains and the interior surface of the pore, which is independent of ionic strength. For the long events, values of Π decrease exponentially with polymer mass, according to the scaling law of Daoud and de Gennes. For PEG molecules larger than ∼5 kDa, Π reached a limiting value suggesting that these PEG chains cannot fit entirely into the β-barrel

Subunit composition of a bicomponent toxin: Staphylococcal leukocidin forms an octameric transmembrane pore

Staphylococcal leukocidin pores are formed by the obligatory interaction of two distinct polypeptides, one of class F and one of class S, making them unique in the family of β-barrel pore-forming toxins (β-PFTs). By contrast, other β-PFTs form homo-oligomeric pores; for example, the staphylococcal α-hemolysin (αHL) pore is a homoheptamer. Here, we deduce the subunit composition of a leukocidin pore by two independent methods: gel shift electrophoresis and site-specific chemical modification during single-channel recording. Four LukF and four LukS subunits coassemble to form an octamer. This result in part explains properties of the leukocidin pore, such as its high conductance compared to the αHL pore. It is also pertinent to the mechanism of assembly of β-PFT pores and suggests new possibilities for engineering these proteins