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.

Background: Occ channels mediate small molecule uptake in Pseudomonads. Results: We have analyzed a number of site-directed mutants for two Occ channels. Conclusion: Pores of OccD subfamily members are highly flexible. The central basic ladder residues interact with the substrate carboxyl group and are essential for transport. Significance: The data provide the first atomistic insights into transport by an important class of OM channels.

Pseudomonas aeruginosa is a Gram-negative bacterium that utilizes substrate-specific outer membrane (OM) proteins for the uptake of small, water-soluble nutrients employed in the growth and function of the cell. In this paper, we present for the first time a comprehensive single-channel examination of seven members of the OM carboxylate channel K (OccK) subfamily. Recent biochemical, functional and structural characterization of the OccK proteins revealed their common features, such as a closely related, monomeric, 18-stranded β-barrel conformation with a kidney-shaped transmembrane pore and the presence of a basic ladder within the channel lumen. Here, we report that the OccK proteins exhibited fairly distinct unitary conductance values, in a much broader range than earlier expectations, which includes low (~40–100 pS) and medium (~100–380 pS) conductance. These proteins showed diverse single-channel dynamics of current gating transitions, revealing one (OccK3)-, two (OccK4, OccK5 and OccK6)- and three (OccK1, OccK2 and OccK7)-open sub-state kinetics with functionally distinct conformations. Interestingly, we discovered that anion selectivity is a conserved trait among the members of the OccK subfamily, confirming the presence of a net pool of positively charged residues within their central constriction. Moreover, these results are in accord with an increased specificity and selectivity of these protein channels for negatively charged, carboxylate-containing substrates. Our findings might ignite future functional examinations and full-atomistic computational studies for unraveling a mechanistic understanding of the passage of small molecules across the lumen of substrate-specific, β-barrel OM proteins.

OpdK is an outer membrane protein of the pathogenic bacterium Pseudomonas aeruginosa. The recent crystal structure of this protein revealed a monomeric, 18-stranded β-barrel with a kidney-shaped pore, whose constriction features a diameter of 8 Å. Using systematic single-channel electrical recordings of this protein pore reconstituted into planar lipid bilayers under a broad range of ion concentrations, we were able to probe its discrete gating kinetics involving three major and functionally distinct conformations, in which a dominant open sub-state O2 is accompanied by less thermodynamically stable sub-states O1 and O3. Single-channel electrical data enabled us to determine the alterations in the energetics and kinetics of the OpdK protein when experimental conditions were changed. In the future, such a semi-quantitative analysis might provide a better understanding on the dynamics of current fluctuations of other β-barrel membrane protein channels.

β-barrel membrane proteins often fluctuate among various open sub-states, yet the nature of these transitions is not fully understood. Using temperature-dependent, single-molecule electrophysiology analysis, along with rational protein design, we show that OccK1, a member of the outer membrane carboxylate channel from Pseudomonas aeruginosa, features a discrete gating dynamics comprising of both enthalpy-driven and entropy-driven current transitions. OccK1 was chosen to analyze these transitions, because it is a monomeric transmembrane β-barrel of known high-resolution crystal structure and displays three distinguishable, time-resolvable open sub-states. Native and loop-deletion OccK1 proteins showed substantial changes in the activation enthalpies and entropies of the channel transitions, but modest alterations in the equilibrium free energies, confirming that the system never departs from equilibrium. Moreover, some current fluctuations of OccK1 indicated a counterintuitive, negative activation enthalpy, which was compensated by a significant decrease in the activation entropy. Temperature scanning of the single-channel properties of OccK1 exhibited a thermally-induced switch of the energetically most favorable open sub-state at the lowest examined temperature of 4°C. Therefore, such a semi-quantitative assessment of the current fluctuation dynamics not only demonstrates the complexity of channel gating, but also reveals distinct functional traits of a β-barrel outer membrane protein under different temperature circumstances.