Browsing by Author "Ryan, Matthew J."

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    Do selectivity filter carbonyls in K+ channels flip away from the pore? Two-dimensional infrared spectroscopy study
    (Journal of Structural Biology: X, 2024-07-27) Maroli, Nikhil; Ryan, Matthew J.; Zanni, Martin T.; Kananenka, Alexei A.
    Highlights • This work makes a critical contribution towards understanding the structure of K+ channels. • We use computational two-dimensional infrared (2D IR) spectroscopy to design an experiment that would test the existence of a selectivity filter configuration in which one of the Val carbonyls flips away from the pore. • This configuration was hypothesized to be relevant for C-type inactivation of K+ channels. • Existing structural biology tools could not provide a decisive evidence for Val flipped configuration. • Our paper shows that 2D IR spectroscopy is capable of detecting such a configuration. Abstract Molecular dynamics simulations revealed that the carbonyls of the Val residue in the conserved selectivity filter sequence TVGTG of potassium ion channels can flip away from the pore to form hydrogen bonds with the network of water molecules residing behind the selectivity filter. Such a configuration has been proposed to be relevant for C-type inactivation. Experimentally, X-ray crystallography of the KcsA channel admits the possibility that the Val carbonyls can flip, but it cannot decisively confirm the existence of such a configuration. In this study, we combined molecular dynamics simulations and line shape theory to design two-dimensional infrared spectroscopy experiments that can corroborate the existence of the selectivity filter configuration with flipped Val carbonyls. This ability to distinguish between flipped and unflipped carbonyls is based on the varying strength of the electric field inside and outside the pore, which is directly linked to carbonyl stretching frequencies that can be resolved using infrared spectroscopy. Graphical abstract available at: https://doi.org/10.1016/j.yjsbx.2024.100108
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    Probing Ion Configurations in the KcsA Selectivity Filter with Single-Isotope Labels and 2D IR Spectroscopy
    (Journal of the American Chemical Society, 2023-08-23) Ryan, Matthew J.; Gao, Lujia; Valiyaveetil, Francis I.; Zanni, Martin T.; Kananenka, Alexei A.
    The potassium ion (K+) configurations of the selectivity filter of the KcsA ion channel protein are investigated with two-dimensional infrared (2D IR) spectroscopy of amide I vibrations. Single 13C–18O isotope labels are used, for the first time, to selectively probe the S1/S2 or S2/S3 binding sites in the selectivity filter. These binding sites have the largest differences in ion occupancy in two competing K+ transport mechanisms: soft-knock and hard-knock. According to the former, water molecules alternate between K+ ions in the selectivity filter while the latter assumes that K+ ions occupy the adjacent sites. Molecular dynamics simulations and computational spectroscopy are employed to interpret experimental 2D IR spectra. We find that in the closed conductive state of the KcsA channel, K+ ions do not occupy adjacent binding sites. The experimental data is consistent with simulated 2D IR spectra of soft-knock ion configurations. In contrast, the simulated spectra for the hard-knock ion configurations do not reproduce the experimental results. 2D IR spectra of the hard-knock mechanism have lower frequencies, homogeneous 2D lineshapes, and multiple peaks. In contrast, ion configurations of the soft-knock model produce 2D IR spectra with a single peak at a higher frequency and inhomogeneous lineshape. We conclude that under equilibrium conditions, in the absence of transmembrane voltage, both water and K+ ions occupy the selectivity filter of the KcsA channel in the closed conductive state. The ion configuration is central to the mechanism of ion transport through potassium channels.