An Outer Membrane Protein
Undergoes Enthalpy- and
Entropy-Driven Transitions
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Abstract
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 <i>Pseudomonas aeruginosa</i>, 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