An outer membrane protein undergoes enthalpy- and entropy-driven transitions.

β-Barrel membrane proteins often fluctuate among various open substates, 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 both enthalpy-driven and entropy-driven current transitions. OccK1 was chosen for the analysis of these transitions, because it is a monomeric transmembrane β-barrel of a known high-resolution crystal structure and displays three distinguishable, time-resolvable open substates. 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 substate at the lowest examined temperature of 4 °C. Therefore, such a semiquantitative 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.