¹H‐Detected Solid‐State NMR Studies of Water‐Inaccessible Proteins In Vitro and In Situ

Abstract

¹ H detection can significantly improve solid-state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for ¹ H detection is the introduction of exchangeable protons in otherwise deuterated proteins, which has thus far significantly hampered studies of partly water-inaccessible proteins, such as membrane proteins. Herein, we present an approach that enables high-resolution ¹ H-detected solid-state NMR (ssNMR) studies of water-inaccessible proteins, and that even works in highly complex environments such as cellular surfaces. In particular, the method was applied to study the K⁺ channel KcsA in liposomes and in situ in native bacterial cell membranes. We used our data for a dynamic analysis, and we show that the selectivity filter, which is responsible for ion conduction and highly conserved in K⁺ channels, undergoes pronounced molecular motion. We expect this approach to open new avenues for biomolecular ssNMR.