Studying the molecular determinants of potassium channel structure and function in membranes by solid-state NMR

Abstract

Solid-state Nuclear Magnetic Resonance (ssNMR) has made remarkable progress in the structural characterization of membrane proteins systems at atomic resolution. Such studies can be further aided by the use of molecular dynamic simulations. Moreover, ssNMR data can be directly compared to functional studies as ssNMR can be applied to native-like preparations, cell extracts and whole cells. In practice, ssNMR experiments can be still be challenging due to limitations in signal to noise. To overcome such issues, Dynamic Nuclear Polarization (DNP) has become a powerful method to enhance spectroscopic sensitivity by transferring polarization from electrons to nuclei. This thesis shows how the combination of (DNP enhanced) spectroscopic, computational, and functional studies can be used to answer unresolved structure-function questions in the context of the potassium channel KcsA. In Chapter 2, we show that the interaction between the K+ channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K+ channel pore. The turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K+ channel inactivation and exhibits a remarkable structural plasticity that correlates to K+ channel inactivation. In Chapter 3, specific lipid binding to the pore domain of KcsA and chimeric KcsA-Kv1.3 was investigated on the structural and functional level. Our findings elucidate how protein–lipid and protein–protein interactions modulate K+ channel activity. Using a combination of proton-detected ssNMR and long molecular dynamics simulations in chapter 4, we demonstrate that buried water molecules are spread all along the rear of the inactivated filter. In contrast, the same region of the structure appears to be dewetted when the selectivity filter is in the conductive state. We demonstrate the presence of a pathway that allows for the interchange of buried and bulk water, as required for a functional influence of buried water on recovery and slow inactivation. In Chapter 5, we for the first time could directly examine an open-conductive conformation of KcsA by tuning the lipid environment. For CL-rich lipid bilayers, we observed a 40% PO, which was the most negatively charged lipid in our study. Besides stabilizing the turret region connecting the outer transmembrane helix, at the intra cellular side the activation gate was partially open. In chapter 6, we investigated the use of MAS DNP instrumentation operating at 800 MHz/527 GHz as a powerful method to enhance spectroscopic sensitivity for biomolecular ssNMR. We utilized KcsA in presence of the biradical AMUPol or TOTAPOL. This approach allowed us to refine the channel structure and revealed conformational sub-states that are present during two different stages of the channel gating cycle. In detail, our experiments identified specific selectivity filter residues that exhibit conformational flexibility before and after inactivation. Chapter 7 demonstrated that DNP can be established by creating local spin clusters via site-directed spin labeling of KcsA. By spin-labeling KcsA at a cysteine point-mutation with radical, we examined the structural information from PRE and enhance significantly by DNP effect.