Kinetic and Energetic Insights into the Gating of a Single Protein Channel

Abstract

Gating of a beta-barrel membrane protein is a fundamentally important and ubiquitous process in membrane biology. Here, we systematically examined details of the kinetics and energetics of the current fluctuations within the OccK1 protein, a family member of outer membrane carboxylate channels from Pseudomonsa Aeruginosa. Temperature-dependent, single-molecule electrophysiology analysis of the native and loop-deletion OccK1 proteins demonstrated the distinctive nature of two energetic barriers: an enthalpic barrier of the large-current amplitude, infrequent O1 to O2 transitions and an entropic barrier of the low-current amplitude, highly frequent O2 to O3 transitions. Changes in temperature produced asymmetric kinetic and energetic modifications that caused a switch of the most probable open sub-state, O2 to O3, at the lowest examined temperature of 4 0C. Our approach might be used in the future to obtain a semi-quantitative assessment of the discrete fluctuation dynamics in other beta-barrel protein channels of the outer membranes of Gram-negative bacteria, mitochondria and chloroplasts.Acknowledgments: This work is funded in part by grants from the US National Science Foundation (DMR-1006332, L.M.) and the National Institutes of Health (R01 GM088403, L.M. and R01 GM085785, B.v.d.).