A New Outlook Into The Kinetics And Thermodynamics Of A Protein Pore

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)




Liviu Movileanu


enthalpy-entropy compensation;OccK1;Single-channel electrical recordings;Single-molecule biophysics;Spontaneous gating;The kinetic rate theory

Subject Categories

Physical Sciences and Mathematics | Physics


OccK1 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 OccK1 protein when experimental conditions were changed. Temperature-dependent, single-molecule electrophysiology analysis of OccK1, along with rational protein design also features a discrete gating dynamics comprising of both enthalpy-driven and entropy-driven current transitions. 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. 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 4oC. 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.


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