Two-dimensional infrared (2D IR) spectroscopy for elucidating ion occupancies in the selectivity filter of ion channels1.

This article reviews work presented at the ESCBM 2017 on using two-dimensional infrared (2D IR) spectroscopy to probe ion binding configurations in the potassium ion channel KcsA. We discuss two studies in which we use 2D IR spectroscopy in conjunction with protein semisynthesis and molecular dynamics (MD) simulations to test against two competing models of ion permeation and to investigate how changes in the conformation of the intracellular gate affect the structure and ion affinities of the selectivity filter. In our initial study, the 2D IR spectrum of an isotopically labeled KcsA filter reveals two spectral features that correspond to different structures and ion binding configurations. MD simulations help us link the experiments to atomistic structures, allowing us to determine the prevailing mechanism of ion conduction. In a follow-up study, we probe how ion occupancies in the filter change with the opening and closing of the intracellular gate. We experimentally modulate the conformation of KcsA using different sample conditions, and are able to show that the ion occupancies in the filter change with the state of the gate, revealing complexities in the conformational landscape of the potassium ion channel. Our work shows the potential of these techniques in addressing the fundamental biophysical questions in membrane protein structure and function. [ABSTRACT FROM AUTHOR]