Abstract
Conformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. However, correlation between functional and conformational dynamics remained difficult, lacking experimental techniques to monitor sub-millisecond conformational changes. Here, we use the outer membrane protein G (OmpG) as a model system where loop-6 opens and closes the β-barrel pore like a lid in a pH-dependent manner. Functionally, single-channel electrophysiology shows that while closed states are favored at acidic pH and open states are favored at physiological pH, both states coexist and rapidly interchange in all conditions. Using HS-AFM height spectroscopy (HS-AFM-HS), we monitor sub-millisecond loop-6 conformational dynamics, and compare them to the functional dynamics from single-channel recordings, while MD simulations provide atomistic details and energy landscapes of the pH-dependent loop-6 fluctuations. HS-AFM-HS offers new opportunities to analyze conformational dynamics at timescales of domain and loop fluctuations.
Highlights
Conformational changes in ion channels lead to gating of an ion-conductive pore
We previously introduced HS-AFM height spectroscopy (HS-AFM-HS), where the HS-AFM probe is placed at a fixed x, y-position on the sample, and the height fluctuations in the z-direction under the AFM probe are monitored with angstrom precision and 10-μs temporal resolution[18]
We reconstituted outer membrane protein G (OmpG) into POPE/POPG (80:20) lipids at lipid-to-protein ratios (LPR) between 0.5 and 0.7 (w:w; in our case to 0.65 mM lipid/ 0.03 mM protein and 0.92 mM lipid/0.03 mM protein)
Summary
Conformational changes in ion channels lead to gating of an ion-conductive pore. Ion flux has been measured with high temporal resolution by single-channel electrophysiology for decades. OmpG is imbued with both structural and functional simplicity, where the motions of a single loop are supposed to directly open and close the ion conducting pore, transitions which can be characterized with single-channel recordings. The structural characterizations by X-ray crystallography[23] and conventional AFM24 provided static snapshots of the most probable state under a given condition, while NMR reported loop flexibility[20,25], and electrophysiology showed that open and closed states coexisted in all conditions[19,21,22]. A direct correlation between structural conformations and channel functional states was not possible because of the lack of a time-resolved single-molecule structural technique to cover timescales similar to those of electrophysiology measurements
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