1H-Detected Solid-State NMR Studies of Water-Inaccessible Proteins InVitro and InSitu

H-1 detection can significantly improve solid-state NMR spectral sensitivity and thereby allows studying more complex proteins. However, the common prerequisite for (1)Hdetection 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 H-1-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 insitu 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.